ประชุมกรรมการบริหารภาควิชาสัตวแพทยสาธารณสุข ครั้งที่ 1/2564

รายงานการประชมุ กรรมการบรหิ ารภาควชิ าสตั วแพทยสาธารณสขุ
คณะสตั วแพทยศาสตร์ จฬุ าลงกรณ์มหาวทิ ยาลัย
ครงั้ ท่ี 2/2563 วนั ที่ 27 สงิ หาคม 2563
------------------------------------------------------------

กรรมการผเู้ ข้าร่วมประชมุ

1. ศาสตราจารย์ สพ.ญ.ดร. รุง่ ทพิ ย์ ชวนช่นื ประธานกรรมการ
กรรมการ
2. ศาสตราจารย์ น.สพ.ดร. อลงกร อมรศลิ ป์ กรรมการ
กรรมการ
3. อาจารย์ น.สพ.ดร. ธราดล เหลืองทองคา กรรมการ
กรรมการ
4. อาจารย์ สพ.ญ.ดร. สหฤทัย เจียมศรีพงษ์ กรรมการและเลขานกุ าร

5. อาจารย์ น.สพ.ดร. สิรวิทย์ ภักดีพาณชิ ย์กจิ ติดราชการ

6. นางสาวสวุ ณี เฉลิมชยั นุกลู

7. นางสาวพรทิพย์ เส็งสาเรงิ

กรรมการผไู้ ม่เข้าประชมุ
1. รองศาสตราจารย์ น.สพ.ดร. ศุภชยั เน้ือนวลสุวรรณ

ประธานเปิดการประชุมและเร่ิมประชมุ เวลา 12.00 น.

วาระท่ี 1 เรื่องรบั รองรายงานการประชมุ คร้ังที่ 1/2563
ประธานฯ เสนอท่ปี ระชุมพจิ ารณารับรองรายงานการประชมุ ครงั้ ท่ี 1/2563 วันที่ 24 เมษายน 2563

ทปี่ ระชมุ พิจารณาแล้วมีมติรับรองรายงานการประชมุ

วาระที่ 2 เรื่องแจ้งเพอื่ ทราบ
2.1 ประธานฯ แจง้ ตอ่ ทป่ี ระชุมเรอื่ ง การประชุมวชิ าการ CUVC2020 Live ซ่ึงกาหนดจะจัดงานประชมุ

ในวันที่ 8 – 9 ตุลาคม 2563 (Online) โดยภาควิชาฯ ได้ส่งแบบฟอร์มแจ้งรายชอ่ื คณาจารย์ และบคุ ลากรผู้เข้าร่วม
ประชุมไปยังฝ่ายวจิ ัยเรียบรอ้ ยแล้ว

2.2 ประธานฯ แจง้ ตอ่ ที่ประชุมเร่อื ง รางวัลการวจิ ัย กองทุนรัชดาภเิ ษกสมโภช ประจาปี พ.ศ. 2563
โดยทางมหาวิทยาลยั ขอเชิญผู้สนใจส่งใบสมัครเพ่อื ขอรบั พิจารณารางวัลการวจิ ยั ซ่ึงประกอบดว้ ย

2.2.1 รางวลั ผลงานวิจัยสาหรบั บคุ ลากรประจาของจฬุ าลงกรณม์ หาวิทยาลัย
2.2.2 รางวัลผลงานวิจัยสาหรับนิสติ ระดับปริญญาเอกและปริญญาโทของจุฬาลงกรณ์มหาวทิ ยาลัย

2.3 ประธานฯ แจง้ ตอ่ ที่ประชุมเร่ือง การ Roadshow ของรองอธกิ ารบดี (ศาสตราจารย์ ดร. จักรพนั ธ์
สทุ ธริ ัตน)์ เพอื่ จะชแ้ี จงแนวทางการวิจยั กรอบการวจิ ัย ใหแ้ กค่ ณาจารยแ์ ละบุคลากรของคณะสัตวแพทยศาสตร์
ในวันท่ี 3 กนั ยายน 2563 เวลา 10.00 น. ณ หอ้ งประชุม 4 ตึก 50 ปี

-2-

วาระที่ 3 เรอื่ งเสนอเพือ่ พิจารณา
3.1 ประธานฯ เสนอทปี่ ระชมุ เร่ือง การเปิดรบั สมคั รตาแหน่งอาจารยใ์ หม่ แทน อาจารย์ น.สพ. ทวศี ักดิ์

เจนธนกิจ ซ่งึ ขอลาออกจากตาแหน่งอาจารย์ A-5 ภาควิชาสตั วแพทยสาธารณสขุ แล้ว เมอ่ื วันท่ี 31 กรกฎาคม 2563
โดยเรื่องการลาออกดังกลา่ วได้ผ่านมติที่ประชุมกรรมการประจาคณะสตั วแพทยศาสตร์ เม่อื วนั ท่ี 26 สิงหาคม 2563
ตอ่ จากนีภ้ าควชิ าฯ ต้องดาเนินการเปดิ รบั สมคั ร และกาหนดคุณสมบตั ผิ ู้สมัคร ทั้งนีท้ ปี่ ระชุมพจิ ารณาแลว้ มีมติให้
นางสาวพรทิพย์ เสง็ สาเริง ทาบนั ทึกข้อความเรอื่ งการขอเปดิ รบั สมัคร ตาแหนง่ อาจารย์ A-5 และขอแตง่ ตง้ั
คณะกรรมการดาเนนิ การสรรหาไปยงั ฝา่ ยบุคคลเพ่ือดาเนินการต่อไป

3.2 ประธานฯ เสนอทีป่ ระชุมเร่อื ง การเปิดรับสมคั รพนักงานมหาวทิ ยาลัย (หมวดเงนิ อดุ หนุน) ตาแหน่ง
เจ้าหนา้ ท่ีบรกิ ารวิทยาศาสตร์ (นกั วทิ ยาศาสตร์ P7) แทน คณุ ไฉไล ควู ัฒนานุกูล ซงึ่ ได้เกษยี ณอายรุ าชการเมื่อวนั ที่
30 กันยายน 2562 โดยภาควิชาฯ ได้ทาบันทกึ ขอ้ ความไปยังฝ่ายบคุ คล เพอ่ื เปดิ รบั สมัครแลว้ เมอ่ื เดอื นกรกฎาคม
จนถึงปัจจุบัน มผี สู้ มคั รท้งั สนิ้ จานวน 7 คน ท้งั นี้ที่ประชุมพิจารณาแลว้ มีมติให้ นางสาวพรทิพย์ เสง็ สาเรงิ ประสาน
กับฝ่ายบคุ คล เพือ่ ขอใบสมัครมาพิจารณาคุณสมบตั ิก่อนการสอบคดั เลอื ก

3.3 ประธานฯ เสนอที่ประชุมเรอื่ ง การเกษยี ณอายุราชการของ นางสาวพรทิพย์ เส็งสาเรงิ พนักงาน
มหาวทิ ยาลัย (หมวดเงินอุดหนุน) ตาแหน่งเจ้าหน้าท่สี านกั งาน (บริหารงานทั่วไป P7) ในวนั ที่ 30 กนั ยายน 2563
ภาควิชาฯ ได้ขอคงกรอบอัตราเกษยี ณไปที่มหาวิทยาลัย และได้รับแจง้ ผลการคงกรอบอตั รากลับมายังคณะฯ แลว้
ตอ่ จากนีภ้ าควชิ าฯ ตอ้ งดาเนนิ การเปดิ รับสมัคร และกาหนดคุณสมบัติผ้สู มัคร ท้ังน้ีทป่ี ระชมุ พจิ ารณาแล้วมีมติให้
นางสาวพรทิพย์ เส็งสาเริง ทาบันทึกข้อความเรือ่ งการขอเปดิ รบั สมัครตาแหนง่ เจา้ หนา้ ที่สานกั งาน (บรหิ ารงาน
ท่ัวไป P7) และขอแตง่ ต้งั คณะกรรมการดาเนินการสรรหาไปยังฝ่ายบคุ คลเพ่ือดาเนนิ การต่อไป

วาระท่ี 4 เรอื่ งอ่นื ๆ -ไมม่ -ี

ปิดประชมุ เวลา 14.00 น.
นางสาวพรทพิ ย์ เสง็ สาเรงิ
ผ้บู ันทึกรายงานการประชมุ

บันทึกขอ ความ

สวนงาน กลมุ ภารกจิ สนบั สนุนบณั ฑิตศกึ ษา ฝายวิชาการ บัณฑติ วิทยาลยั โทร. 0-2218-3502-3 โทรสาร 0-2218-3504

ท่ี อว 64.25/ วนั ที่

เร่อื ง การผอนผันการมคี ะแนนภาษาองั กฤษแกนสิ ติ ทีส่ มคั รขอรับทนุ การศกึ ษา ปการศกึ ษา 2564

ในสถานการณท ม่ี ผี ลกระทบจากแพรระบาดของเชื้อโรค COVID-19

เรยี น คณบดที กุ คณะ/ผอู ํานวยการหลกั สตู รสหสาขาวิชา

ตามท่ีบณั ฑิตวทิ ยาลัยไดประกาศรับสมัครทุนอุดหนุนการศึกษา ปก ารศึกษา 2564 และผูสมัคร
รับขอทนุ จะตองมีคะแนนภาษาองั กฤษกอนสมคั รขอรบั ทุนการศกึ ษาไปแลวนั้น เนือ่ งจากเกิดจากสถานการณ
การแพรร ะบาดของเชอื้ ไวรสั COVID-19

คณะกรรมการบริหารบัณฑิตวิทยาลัยในการประชุมครั้งท่ี 8/2564 วันที่ 19 สิงหาคม 2564
มีมติเห็นชอบใหบัณฑิตวิทยาลัยขยายเวลาการยกเวนคะแนนภาษาอังกฤษใหผูรับทุนอุดหนุนการศึกษา
ปการศึกษา 2564 โดยมีเงอื่ นไขดงั น้ี

1. ผูรับทุนภาคการศึกษาตน ปการศึกษา 2564 จะตองมีคะแนนภาษาอังกฤษ ภายในภาค
การศึกษาปลาย ปก ารศึกษา 2564

2. ผูสมัครขอรับทุนภาคการศึกษาปลาย ปการศึกษา 2564 จะตองมีคะแนนภาษาอังกฤษ
ภายในภาคการศกึ ษาตน ปการศึกษา 2565

3. กรณีไมส ามารถดําเนินการไดภายใน 1 ปการศกึ ษาใหพนสถานภาพจากผูรบั ทนุ

จงึ เรยี นมาเพ่อื โปรดทราบ และดาํ เนินการตอไปดว ย

(รองศาสตราจารย ดร.ธรรมนญู หนจู กั ร)
คณบดีบณั ฑติ วทิ ยาลัย

บนั ทกึ ข้อความ

ส่วนงาน กลมุ่ ภารกิจสนับสนุนบณั ฑิตศกึ ษา ฝา่ ยวิชาการ บัณฑิตวิทยาลัย โทร. 83502-3 โทรสาร 83504

ท่ี อว 64.25/ วันที่ สงิ หาคม 2564

เร่อื ง ผลการพจิ ารณาทุนอุดหนนุ การศกึ ษาเฉพาะค่าเล่าเรียนประเภท 60/40 แก่นิสิตท่ีไดร้ ับผลกระทบจากสถานการณ์

แพร่ระบาดของเช้ือโรค COVID-19 ภาคการศึกษาตน้ ปีการศึกษา 2564 รอบที่ 1

เรยี น คณบดี

ตามท่ีคณะได้ส่งรายช่ือผู้ขอรับทุนอุดหนุนการศึกษาเฉพาะค่าเล่าเรียนประเภท 60/40 แก่นิสิตที่ได้รับ
ผลกระทบจากสถานการณ์แพร่ระบาดของเช้ือโรค COVID-19 ภาคการศึกษาต้น ปีการศึกษา 2564 ให้บัณฑิตวิทยาลัย
พิจารณานั้น คณะกรรมการบรหิ ารบณั ฑิตวิทยาลยั ในการประชุม ครง้ั ที่ 8/2564 วันที่ 19 สิงหาคม 2564 มีมติอนุมัติ
ผู้ได้รับทุน จานวน 115 คน (ตามประกาศแนบ) จึงขอให้คณะแจ้งนิสิตผู้ได้รับทุนส่งแบบขอเบิกเงินทุนพร้อม
ใบเสร็จรับเงินการชาระเงินค่าเล่าเรยี น ทางเมลล์ [email protected] และเม่ือบัณฑิตวิทยาลัยเปิดทาการปกติ
ให้ส่งข้อตกลงฉบับจริงท่ีหน่วย One stop service บัณฑิตวิทยาลัย โดยให้ดาวโหลดแบบฟอร์มขอเบิกเงินได้ท่ี
เวบ็ ไซต์ https://www.grad.chula.ac.th (เมนทู ุนอุดหนุนการศึกษา)

จึงเรียนมาเพื่อโปรดดาเนินการ จกั ขอบคุณยง่ิ

(รองศาสตราจารย์ ดร.ธรรมนญู หนูจักร)
คณบดีบณั ฑติ วทิ ยาลยั

ประกาศ
บณั ฑิตวิทยาลัย จฬุ าลงกรณม์ หาวิทยาลัย
เรือ่ ง ผลการพิจารณาทุนอดุ หนนุ การศึกษาเฉพาะคา่ เล่าเรียนประเภท 60/40
แก่นสิ ติ ที่ได้รบั ผลกระทบจากสถานการณแ์ พร่ระบาดของเช้ือโรค COVID-19
ภาคการศกึ ษาต้น ปีการศกึ ษา 2564 (ครั้งท่ี 1)

ตามที่บัณฑิตวิทยาลัยร่วมกับคณะจัดสรรงบประมาณในการสนับสนุนทุนอุดหนุนการศึกษาเฉพาะ
ค่าเล่าเรียนประเภท 60/40 แก่นิสิตท่ีได้รับผลกระทบจากสถานการณ์แพร่ระบาดของเชื้อโรค COVID-19
ในภาคการศึกษาต้น ปีการศึกษา 2564 เพ่ือช่วยเหลือนิสิตระดับบัณฑิตศึกษา โดยเป็นค่าเล่าเรียนตามจานวนท่ีจ่ายจริง
(ในอัตรานิสิตชาวไทยไม่รวมค่าธรรมเนียมการศึกษา) น้ัน คณะกรรมการบริหารบัณฑิตวิทยาลัยในการประชุม
คร้ังท่ี 8/2564 วันท่ี 19 สิงหาคม 2564 มีมติอนมุ ตั ใิ หน้ ิสิตได้รบั ทนุ ดังกลา่ ว จานวน 115 คน ดังนี้

เลขที่ รหัสนสิ ติ ชื่อ-นามสกุล สาขาวชิ า

1) คณะครุศาสตร์ จานวน 9 คน พัฒนศกึ ษา
การศึกษานอกระบบโรงเรยี น
ระดับปริญญาดุษฎีบณั ฑิต การศึกษานอกระบบโรงเรียน
การศึกษานอกระบบโรงเรียน
1. 6184201427 นางกระจ่างศรี ศรีกระจ่าง ผสมทรัพย์
สถิตแิ ละสารสนเทศทางการศึกษา
2. 6184215227 นางสาวบุษบากร สวุ รรณเกษา สถติ แิ ละสารสนเทศทางการศึกษา
สถิตแิ ละสารสนเทศทางการศึกษา
3. 6184216927 นายปรเมธ อดุ มสินานนท์ เทคโนโลยแี ละสื่อสารการศึกษา
เทคโนโลยีและส่ือสารการศึกษา
4. 6184216927 นายปรเมธ อุดมสนิ านนท์

ระดบั ปริญญามหาบัณฑติ

5. 6183404327 นางสาวประกายแก้ว ไกรสงคราม

6. 6183409527 นายยทุ ธชัย ศิลาแลง

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9. 6280039727 นาย ชาฤทธิ์ วริ ยิ ะชาญ

2

เลขท่ี รหัสนิสติ ช่ือ-นามสกุล สาขาวิชา

2) บัณฑิตวิทยาลัย จานวน 15 ทุน

ระดบั ปรญิ ญาดุษฎบี ณั ฑติ

10. 5987828020 นางสาวอาทติ ยา ช่างด้วง การจดั การสารอนั ตรายและส่งิ แวดล้อม

11. 6087852420 นางสาวฐาปนี แสงเพชร การจัดการสารอนั ตรายและสง่ิ แวดลอ้ ม

12. 5987805520 นางสาวสมฤทัย รตั นบุรี ชวี เวชศาสตร์

13. 6187763320 นายชาญศักดิ์ เสยี งเย็น ภาษาองั กฤษเป็นภาษานานาชาติ

14. 6087813020 นายฮซู าม อีทจิ านิ วิทยาศาสตรน์ าโนและเทคโนโลยี

15. 5987758720 นางสาวจรีลกั ษ์ เปลง่ สกลุ วิทยาศาสตรส์ ิ่งแวดล้อม

16. 6087823320 Mr.Xiaoxi Guo สิง่ แวดล้อม การพฒั นาและความยงั่ ยืน

ระดบั ปรญิ ญามหาบัณฑิต

17. 6187582020 นางสาวถริ าภา ภราดรธรรม การจดั การสารอันตรายและสง่ิ แวดล้อม

18. 6187286320 นางสาวปนดั ดา อยอู่ ภิบาลรักษ์ จลุ ชวี วทิ ยาทางการแพทย์

19. 6280039820 นางสาวพรรณพลอย แกลว้ กล้า จลุ ชีววทิ ยาทางการแพทย์

20. 6280071820 นางสาวอภิชญา ศรีวะรมย์ จลุ ชีววทิ ยาทางการแพทย์

21. 6280101020 นางสาวรกั วารี สหี ราช จุลชีววิทยาทางการแพทย์

22. 6280078220 นางสาวขวัญฤดี สุวรรณสงั ข์ วิทยาศาสตร์ส่ิงแวดลอ้ ม

23. 6280088520 นางสาวศราวรรณ หงษ์วเิ ศษ วิทยาศาสตร์ส่ิงแวดลอ้ ม

24. 6280090720 นางสาวอจั ฉรยี า สัมพันธ์พร วทิ ยาศาสตรส์ ิง่ แวดล้อม

3) คณะแพทยศาสตร์ 4 ทุน

ระดบั ปรญิ ญามหาบณั ฑติ

25. 6174034330 นางสาวอรนรี เชาวน์ ะรัง เวชศาสตร์การกีฬา

26. 6174035030 นายอาทติ ย์ งามช่ืน เวชศาสตรก์ ารกีฬา

27. 6270014030 นางสาวปียานฐิ ธนวนชิ นาม เวชศาสตรก์ ารกฬี า

28. 6270016330 นายวรทย์ รังสมิ าหริวงศ์ เวชศาสตร์การกีฬา

4) คณะวทิ ยาศาสตร์การกีฬา จานวน 1 ทุน

ระดับปริญญามหาบัณฑิต

29. 6178304539 นายธเนศ จนิ ดา วิทยาศาสตรก์ ารกฬี า

3

เลขท่ี รหสั นสิ ิต ช่อื -นามสกุล สาขาวชิ า

5) คณะวศิ วกรรมศาสตร์ จานวน 45 ทนุ

ระดับปรญิ ญาดุษฎีบณั ฑติ

30. 5971464421 นางสาวนริตา จนั ทร์ทนต์ วศิ วกรรมเคมี

31. 6071413921 นายชุตวิ ัต อรรถบรู ณว์ งศ์ วิศวกรรมเคมี

32. 6071416821 นายณัฐวัตร เพชรสูงสกลุ วิศวกรรมเคมี

33. 6171431221 MRS.ASDARINA YAHYA วศิ วกรรมเคมี

34. 6171437021 นายคุณวัฒน์ อนุ ทักษณิ กลุ วศิ วกรรมเคมี

35. 6171448021 นายเจษฎา ปริตาโพธ์ิ วศิ วกรรมโยธา

ระดบั ปรญิ ญามหาบณั ฑติ

36. 6170354421 นายเจตนพิ ฐิ อรณุ รัตน์ วิศวกรรมเครื่องกล

37. 6170361821 นายชยั วฒั น์ มุสิกะปาน วิศวกรรมเครื่องกล

38. 6170379121 นายนธิ ิชยั คาดการณ์ไกล วศิ วกรรมเครื่องกล

39. 6270089821 นายณัฐภทั ร คิว้ วงศง์ าม วิศวกรรมเครื่องกล

40. 6270344821 นางสาวฐติ ิพร อ้ายดี วิศวกรรมเครื่องกล

41. 6270367221 นางสาวเกวลี อัศวะไพฑูรย์เสรฐิ วิศวกรรมเคร่อื งกล

42. 6270372321 นายชยธร ก้เู กียรติกูล วศิ วกรรมเครื่องกล

43. 6070144921 นายจฑุ านนท์ บุญประเสรฐิ วศิ วกรรมโลหการ

44. 6070300421 นายวรทตั หงษท์ อง วิศวกรรมไฟฟ้า

45. 6170210721 นางสาวปรยี ฉตั ร จักษุดลุ ย์ วศิ วกรรมเคมี

46. 6170312021 นางสาวสุภัสสร จอกแกว้ วิศวกรรมสิ่งแวดลอ้ ม

47. 6170420121 นายณฐั พพิ ัฒน์ ฤกษ์รุ่งจริยา วศิ วกรรมสิ่งแวดลอ้ ม

48. 6170428221 นางสาวอัมพรรชั ภูสุวรรณ์ วศิ วกรรมสง่ิ แวดล้อม

49. 6170438521 นางสาวครองขวญั ข่วงบุญ วิศวกรรมสง่ิ แวดล้อม

50. 6170444221 นายชินวตั ร ชะนะปาโมกโข วศิ วกรรมสง่ิ แวดล้อม

51. 6170460221 นายเบญจพล เบญจวรางกลู วิศวกรรมโยธา

52. 6170496921 นางสาวองิ ฉตั ร สุภาพยาม วิศวกรรมสิง่ แวดล้อม

53. 6270007321 นายกฤตภาส สุวรรณมณี วศิ วกรรมสง่ิ แวดลอ้ ม

54. 6270031321 นางสาวจรสั อรณุ เจรญิ สวัสด์ิ วศิ วกรรมสิง่ แวดล้อม

55. 6270061121 นางสาวชัญญานชุ วชิราศรีศิรกิ ลุ วิศวกรรมสง่ิ แวดลอ้ ม

56. 6270063421 นายชัยพทั ธ์ วิสารทกลุ วิศวกรรมสง่ิ แวดลอ้ ม

4

เลขท่ี รหัสนสิ ติ ชอื่ -นามสกุล สาขาวิชา
วิศวกรรมเคมี
57. 6270064021 นายชยั วรรณ กอบางยาง วิศวกรรมส่ิงแวดลอ้ ม
วิศวกรรมสิง่ แวดลอ้ ม
58. 6270159021 นางสาวปวนั รัตน์ สาลีผล วิศวกรรมสิ่งแวดล้อม
วศิ วกรรมเคมี
59. 6270227021 นางสาวรสสุคนธ์ สิงห์สม วศิ วกรรมโยธา
วศิ วกรรมเคมี
60. 6270246021 นางสาววรรณพศกุ ร์ สทิ ธผิ ล วศิ วกรรมสงิ่ แวดล้อม
วิศวกรรมเคมี
61. 6270253321 นางสาววรศิ รา ไพศาลธรรม วิศวกรรมและเทคโนโลยีการป้องกันประเทศ
วิศวกรรมสง่ิ แวดล้อม
62. 6270277421 นายศวิ กร นชุ สวาท วศิ วกรรมสิ่งแวดลอ้ ม
วศิ วกรรมส่งิ แวดลอ้ ม
63. 6270288321 นายสิทธา ตัณฑ์ไพบูลย์ วศิ วกรรมสิ่งแวดลอ้ ม
วศิ วกรรมสิ่งแวดลอ้ ม
64. 6270304721 นางสาวสุภิญญา กลู นรา วิศวกรรมส่ิงแวดลอ้ ม
วศิ วกรรมไฟฟ้า
65. 6270320721 นางสาวอาทิตยา มาลาทพิ ย์ วศิ วกรรมสง่ิ แวดล้อม

66. 6270328821 นายขวัญชยั บุญประดษิ ฐ์ เศรษฐศาสตร์
เศรษฐศาสตร์
67. 6270330021 นายปกรณเ์ กียรติ หมน่ื สิทธโิ รจน์
สถาปัตยกรรม
68. 6270333921 นางสาวรสิตา รืน่ หาญ

69. 6270334521 นางสาวลลติ า หลาพ่ึง

70. 6270337421 นายสวิท วเิ ศษคณุ ธรรม

71. 6270338021 นางสาวสพุ รรณี ธีรวัฒนสาร

72. 6270339721 นางสาวอาภาพร นาภูมิ

73. 6270361421 นางสาววรี ดา หมัดสี

74. 6270383221 นายภาวัต ธนะจิระเดช

6) คณะเศรษฐศาสตร์ จานวน 2 ทุน

ระดับปรญิ ญามหาบัณฑิต

75. 6185170829 นายศิรวธุ สุยาม

76. 6280020229 นายศกั ดิ์ศิริ เสาโกมุท

7) คณะสถาปัตยกรรมศาสตร์ จานวน 1 ทนุ

ระดบั ปรญิ ญามหาบณั ฑติ

77. 6173371625 นางสาวสุภดิ า พลู สขุ โข

5

เลขที่ รหสั นสิ ติ ช่ือ-นามสกุล สาขาวชิ า

8) คณะสหเวชศาสตร์ จานวน 12 ทนุ ชวี เคมีคลินิกและอณูทางการแพทย์

ระดับปรญิ ญาดุษฎบี ณั ฑติ อาหารและโภชนาการ
กายภาพบาบดั
78. 5876667337 นายอัษฎางศ์ ธีระศรี วทิ ยาศาสตรร์ ะดบั โมเลกุลทางจุล
ชีววทิ ยาทางการแพทยแ์ ละวิทยา
ระดับปรญิ ญามหาบณั ฑติ ภมู คิ ุ้มกัน
อาหารและโภชนาการ
79. 6076851037 นายธนะศกั ดิ์ ตันติชัยยกุล ชีวเคมคี ลินิกและอณทู างการแพทย์
กายภาพบาบดั
80. 6176656037 นางสาวซาร่า ดาวเด็ง กายภาพบาบดั
กายภาพบาบัด
81. 6176754937 นางแพรทิพย์ คล้ายเจรญิ สขุ วทิ ยาศาสตร์โลหิตวิทยาคลนิ กิ
ชวี เคมคี ลนิ ิกและอณทู างการแพทย์
82. 6176851037 นายนันท์ธร เจริญศักด์ิ ชีวเคมคี ลินกิ และอณูทางการแพทย์

83. 6270001837 นางสาวกมลวรรณ ชยั คง พยาธชิ วี วทิ ยาทางสตั วแพทย์
พยาธชิ ีววิทยาทางสัตวแพทย์
84. 6270005337 นางสาวนิออน แย้มสงวนศักดิ์ อายรุ ศาสตรส์ ตั วแพทย์
พยาธิชวี วทิ ยาทางสัตวแพทย์
85. 6270009937 นายภคั เนยี มสุวรรณ พยาธิชวี วิทยาทางสตั วแพทย์
พยาธชิ วี วทิ ยาทางสัตวแพทย์
86. 6270010437 นายภาคภูมิ มาสมบรู ณ์ พยาธิชวี วทิ ยาทางสัตวแพทย์
พยาธชิ ีววิทยาทางสตั วแพทย์
87. 6270015637 นางสาวอจั ฉราภรณ์ เจียรนัยกรู
สตั วแพทยสาธารณสขุ
88. 6270017937 นางสาวขวัญจริ า ทรงศรีทยา อายรุ ศาสตรส์ ตั วแพทย์
เภสัชวทิ ยาทางการสตั วแพทยศาสตร์
89. 6270021337 นายวษิ ณุ เนียมสี อายุรศาสตรส์ ัตวแพทย์

9) สัตวแพทยศาสตร์ จานวน 12 ทนุ

ระดบั ปรญิ ญาดุษฎบี ณั ฑติ

90. 5775315131 นายเจษฎา ศริ พิ นู ทรัพย์

91. 5875509731 นางสาวภาวญิ า ภผู า

92. 5875515431 นายกฤษฎา บญุ อรา่ มเรือง

93. 5875520531 นางสาวสุชญา พนั ธ์พฒั นกลุ

94. 5975501731 นางสาวกรรณกิ าร์ พงษ์รปู

95. 5975510331 นายประเสรฐิ อภิวัฒนศ์ ิริ

96. 6175506431 นางสาวเบญจมาศ คุระจอก

97. 6175507031 นางสาวปยิ นชุ ฟองเหม

ระดบั ปริญญามหาบัณฑติ

98. 6175312031 นายสิรวิชญ์ ด่านวนชิ วงศ์

99. 6175320031 นางสาวเพ็ญพชิ ชา เกิดบ้านตะเคียน

100. 6270003431 นายธนวิทย์ สีหอาไพ

101. 6270011431 นางสาวศริ ินันท์ ศรบี ัวสด

6

เลขที่ รหสั นสิ ติ ช่อื -นามสกุล สาขาวชิ า

10) คณะอักษรศาสตร์ จานวน 13 ทุน แสงพันธ์ ภาษาไทย
ระดบั ปรญิ ญาดุษฎบี ัณฑติ
ภาษาอังกฤษ
102. 5880121522 นายณฐั วฒุ ภาษาไทย
ประวัตศิ าสตร์
ระดบั ปรญิ ญามหาบณั ฑิต ครุ ุรตั น์พันธ์ การแปลและการลา่ ม
103. 6080155422 นายศราวธุ สวยบารุง ภาษาองั กฤษ
104. 6180159022 นางสาวรตั นาวดี ปานอืน ภาษาอังกฤษ
105. 6180176722 นายอภิสิทธ์ิ ภาษาอังกฤษ
ภาษาไทย
106. 6180339722 นางสาววรา หาญสิริเศรษฐ ภาษาไทย
ภาษาไทย
107. 6280003722 นายกนั ตพฒั น์ อนพุ นั ธุ์สกลุ ภาษาอังกฤษ
ภาษาไทย
108. 6280007222 นางสาวจตตรุ พร คงบาง

109. 6280010022 นายชยพล ค่าแพง

110. 6280011722 นายชนิ ภทั ร หนูสงค์

111. 6280033522 นายยทุ ธการ ปทั มโรจน์

112. 6280043822 นางสาวศศพิ ิมพ์ ภ่รู ะหงษ์

113. 6280044422 นายศริ วิทย์ ศรีภธู ร

114. 6280051822 นางสาวอภิษฎา ขาวสุด

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ประกาศ ณ วันท่ี สิงหาคม พ.ศ. 2564

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คณบดบี ัณฑิตวิทยาลยั

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ระบบตรีภำค ปกี ำรศึกษำ 2564

ใหก้ ำหนดวนั เปิด – ปดิ ภำคกำรศึกษำของจฬุ ำลงกรณ์มหำวิทยำลยั ระบบตรภี ำค ปีกำรศึกษำ 2564
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ภาคการศกึ ษาทหี่ นึง่

วันเปดิ เรยี นภาคการศึกษาทีห่ นง่ึ วนั จนั ทร์ท่ี 9 สงิ หำคม 2564
วนั สอบกลางภาคการศกึ ษาที่หนงึ่ (ถ้าม)ี วนั จนั ทรท์ ่ี 27 กนั ยำยน - วนั ศกุ รท์ ี่ 1 ตลุ ำคม 2564
วนั สดุ ท้ายของการเรยี นภาคการศกึ ษาท่ีหนงึ่ วันศกุ รท์ ่ี 19 พฤศจิกายน 2564
วนั สอบปลำยภาคการศึกษาที่หน่งึ วันจันทร์ที่ 22 - วนั ศกุ ร์ท่ี 26 พฤศจิกำยน 2564
วันปิดเรยี นภาคการศึกษาทห่ี นงึ่ วันเสาร์ที่ 27 พฤศจิกำยน 2564

ภาคการศกึ ษาท่ีสอง

วันเปิดเรยี นภาคการศึกษาทีส่ อง วนั อังคำรที่ 7 ธนั วาคม 2564
วันสอบกลางภาคการศึกษาท่ีสอง (ถา้ ม)ี วันจนั ทรท์ ี่ 24 - วนั ศุกร์ท่ี 28 มกรำคม 2565
วนั สดุ ท้ายของการเรียนภาคการศึกษาที่สอง วนั ศุกร์ที่ 18 มีนาคม 2565
วันสอบปลำยภาคการศึกษาท่ีสอง วันจนั ทร์ที่ 21 - วนั ศุกร์ท่ี 25 มีนำคม 2565
วันปดิ เรียนภาคการศึกษาที่สอง วนั เสารท์ ี่ 26 มนี าคม 2565

ภาคการศึกษาที่สาม

วันเปดิ เรยี นภาคการศึกษาทสี่ าม วันจันทร์ท่ี 4 เมษายน 2565
วนั สอบกลางภาคการศึกษาที่สาม (ถา้ มี) วันจันทรท์ ่ี 23 - วนั ศุกร์ท่ี 27 พฤษภำคม 2565
วันสดุ ท้ายของการเรยี นภาคการศึกษาท่สี าม วันศกุ ร์ท่ี 15 กรกฎาคม 2565
วนั สอบปลำยภาคการศึกษาท่ีสำม วนั จนั ทร์ที่ 18 - วันศกุ ร์ที่ 22 กรกฎาคม 2565
วันปิดเรียนภาคการศึกษาท่สี าม วันเสำร์ที่ 23 กรกฎาคม 2565
วันสุดทา้ ยของปีการศึกษา วนั ศกุ ร์ที่ 5 สิงหำคม 2565

สัง่ ณ วันที่ เมษำยน พ.ศ. 2564

(ศำสตรำจำรย์ ดร.บณั ฑติ เอื้ออำภรณ์)
อธกิ ำรบดี

คำสง่ั จฬุ ำลงกรณ์มหำวิทยำลัย
ท่ี / 2564

เร่ือง กำหนดวันเปิด – ปิดภำคกำรศึกษำของจุฬำลงกรณม์ หำวิทยำลัย
ระบบทวภิ ำค ปกี ำรศกึ ษำ 2564

ให้กำหนดวนั เปิด – ปิดภำคกำรศกึ ษำของจุฬำลงกรณม์ หำวิทยำลยั ระบบทวิภำค ปกี ำรศึกษำ 2564
ซ่ึงที่ประชุมคณบดีในกำรประชุม คร้ังที่ 6/2564 เมื่อวันที่ 24 มนี ำคม 2564 ไดม้ ีมติเหน็ ชอบแลว้ ดังต่อไปน้ี

ภาคการศกึ ษาต้น

วันเปิดเรยี นภำคกำรศึกษำต้น วนั จันทร์ที่ 9 สงิ หำคม 2564
วันสอบกลำงภำคกำรศึกษำต้น (ถำ้ มี) วนั จันทรท์ ี่ 4 - วนั ศุกร์ท่ี 8 ตุลำคม 2564
วนั ปดิ กิจกรรมภำคกำรศึกษำตน้ วนั อำทิตยท์ ี่ 31 ตุลำคม 2564
วันสุดท้ำยของกำรเรยี นภำคกำรศกึ ษำต้น วนั ศกุ รท์ ี่ 26 พฤศจิกำยน 2564
วนั สอบปลำยภำคกำรศึกษำต้น วันจนั ทรท์ ่ี 29 พฤศจิกำยน – วนั องั คำรที่ 14 ธนั วำคม 2564
วนั ปิดเรียนภำคกำรศึกษำต้น วันพุธท่ี 15 ธันวำคม 2564

ภาคการศึกษาปลาย

วันเปิดเรียนภาคการศึกษาปลาย วนั จนั ทร์ท่ี 10 มกรำคม 2565
วนั สอบกลางภาคการศึกษาปลาย (ถา้ มี) วนั จันทรท์ ี่ 7 - วนั ศุกรท์ ่ี 11 มนี ำคม 2565
วนั ปิดกจกรรมภาคการศึกษาปลาย วันอำทิตยท์ ี่ 3 เมษำยน 2565
วันสดุ ท้ายของการเรียนภาคการศึกษาปลาย วนั ศุกรท์ ่ี 6 พฤษภำคม 2565
วนั สอบปลำยภาคการศึกษาปลำย วันจนั ทร์ที่ 9 - วันอังคำรท่ี 24 พฤษภำคม 2565
วนั ปดิ เรียนภาคการศึกษาปลาย วันพุธท่ี 25 พฤษภำคม 2565

ภาคฤดูร้อน

วนั เปิดเรียนภาคฤดูรอ้ น วันจันทรท์ ี่ 6 มิถนุ ำยน 2565
วนั ศกุ ร์ที่ 22 กรกฎำคม 2565
วันสุดท้ายของการเรยี นและกำรสอบภาคฤดูรอ้ น วนั เสำรท์ ่ี 23 กรกฎำคม 2565
วันศกุ ร์ที่ 5 สิงหำคม 2565
วันปดิ เรยี นภาคฤดูร้อน
วนั สุดทา้ ยของปีการศึกษา

สัง่ ณ วันท่ี เมษำยน พ.ศ. 2564

(ศำสตรำจำรย์ ดร.บัณฑติ เอ้ืออำภรณ์)
อธิกำรบดี

บันทึกขอ้ ความ

ส่วนงาน คณะกรรมการด้านทานุบารุงศิลปวฒั นธรรม และกิจกรรมพเิ ศษ โทร. ๐ ๒๒๑๘ ๙๗๗๖

ท่ี ศว 043 /๒๕๖4 วันท่ี 15 กนั ยายน 2564

เรือ่ ง ขอเรียนเชญิ ร่วมงานเกษยี ณอายุราชการ ประจาปี ๒๕๖4

เรียน หวั หนา้ ภาควชิ า หนว่ ยงาน ........................................................................

ด้วย คณะกรรมการด้านทานุบารุงศลิ ปวฒั นธรรม และกิจกรรมพิเศษ คณะสัตวแพทยศาสตร์

และสมาคมนิสิตเก่าคณะสัตวแพทยศาสตร์ จะจัดงานเกษียณอายุราชการ อาจารย์ บุคลากร ประจาปี ๒๕๖4

ในวันศุกร์ท่ี 8 ตุลาคม ๒๕๖4 ณ อาคาร ๖๐ ปี สัตวแพทยศาสตร์ รูปแบบการจัดงาน นาเสนอประวัติ

ผู้เกษียณอายุราชการทั้ง 9 ท่าน แสดงความรู้สึกจากเพื่อนร่วมงาน มอบโล่ มอบของที่ระลึก แด่ผู้เกษียณอายุ

ราชการ โดยมีอาจารย์ และบคุ ลากร เกษียณอายุราชการอายุครบ 60 ปี จานวน 9 ท่าน และอาจารย์ที่อายคุ รบ

65 ปี จานวน 3 ท่าน ตามกาหนดการดงั ตอ่ ไปนี้

๐๘.๐๐ – ๐๙.0๐ น. ผแู้ ทนผเู้ กษียณอายุราชการทาบญุ ถวายสงั ฆทานฯ

09.00 – 09.30 น. ลงทะเบียน

๐๙.3๐ – ๐๙.40 น. คณบดีกล่าวเปิดงาน

09.40 - 10.00 น. รุ่นพเี่ กษยี ณอายุ 65 ปี กลา่ วตอ้ นรับผู้เกษียณอายรุ าชการ

1. ศาสตราจารย์ น.สพ.ดร.มงคล เตชะกาพุ

2. ศาสตราจารย์ น.สพ.สมชาย จนั ทร์ผอ่ งแสง

3. ศาสตราจารย์ สพ.ญ.ดร.อัจฉริยา ไศละสูต

10.00 – ๑๐.30 น. นาเสนอประวตั ิผเู้ กษียณอายรุ าชการ

๑. ศาสตราจารย์ สพ.ญ.ดร.ชลลดา บรู ณกาล

2. รองศาสตราจารย์ สพ.ญ.ดร.มีนา สาริกะภตู ิ

3. รองศาสตราจารย์ สพ.ญ.ดร.รสมา ภ่สู ุนทรธรรม

4. รองศาสตราจารย์ สพ.ญ.ดร.นนั ทริกา ชนั ซือ่

5. รองศาสตราจารย์ น.สพ.ดร.คณิศักดิ์ อรวีระกุล

๖. นางจงกล แสงวริ ณุ

๗. นางสาววาสนา แสงวารินทร์

8. นายสชุ าติ ทองลิม่

9. นายวเิ ชียร อนชุ นาด

๑๐.30 – ๑๑.15 น. แสดงความร้สู กึ จากเพอ่ื นรว่ มงานถึงผเู้ กษียณอายุราชการ

๑๑.15 – ๑๑.45 น. มอบโล่/มอบของที่ระลกึ

2./ในการน้ี...

-2-
ในการน้ี คณะกรรมการด้านทานุบารุงศิลปวัฒนธรรมและกิจกรรมพิเศษ คณะสัตวแพทยศาสตร์
และสมาคมนิสิตเก่าคณะสัตวแพทยศาสตร์ ขอเรียนเชิญทา่ น และบุคลากรในหน่วยงานเข้าร่วมงาน โดยการสแกน
QR code ด้านทา้ ยจดหมายฉบับนี้ ดังรายละเอยี ดต่อไปน้ี
1. แสดงมทุ ิตาจิตผเู้ กษยี ณอายุราชการ เพอ่ื จัดทาหนังสือท่ีระลกึ งานเกษยี ณฯ
2. ลงทะเบยี นเข้าร่วมงานเกษยี ณอายรุ าชการ
ทั้งนี้ขอความรว่ มมือดาเนนิ การรายการที่ 1 และ 2 ภายในวนั ที่ 28 กนั ยายน 2564
จึงเรียนมาเพอ่ื โปรดทราบ และดาเนินการต่อไปด้วย จะขอบพระคณุ ยิง่

(รองศาสตราจารย์ น.สพ.ดร.นพดล พฬิ ารัตน)์
ผู้ชว่ ยคณบดฝี ่ายกายภาพและศลิ ปวัฒนธรรม

หมายเหตุ : รปู แบบกจิ กรรมอาจมีการเปลย่ี นแปลงตามความเหมาะสมของสถานการณ์โรคระบาด Covid-19



ใบสมคั รงานพนกั งานมหาวิทยาลัยสายวชิ าการ
Academic University Employee
Application Form บคุ ลากรจุฬาฯ ✔ บุคคลท่วั ไป
Internal Candidate External Candidate

ตําแหนง อาจารย

Position

ขอ มลู สว นตัว Personal Information
คํานําหนา : นางสาว ชอ่ื : เบญจวรรณ
นามสกลุ : แซช ือ้

Title : Miss First Name : BENJAWAN Last Name : SAECHUE

วัน/เดอื น/ปเ กิด : 2 สิงหาคม 2535 อายุ : 29 ป สญั ชาติ : ไทย

Date of Birth Age Nationality

เลขท่บี ัตรประชาชน : 1-2009-00133-71-2 เลขทีห่ นังสอื เดินทาง :

ID Number Passport Number

เบอรโ ทรศพั ท : 0847983089 อีเมล : [email protected]

Phone Number E-mail address

สถานภาพทางทหาร : ผานการเกณฑท หาร

Military service status Completed the military services

ไดรบั การผอนผนั /ยกเวน เนอ่ื งจาก

Waiver / Exemption due to

ประวตั ิการศึกษา Education Background

ระดบั การศกึ ษา ประเทศ ชอื่ สถานศกึ ษา วุฒิการศกึ ษา สาขา เกรดเฉล่ีย วนั ทีจ่ บการศึกษา
Education Degree Countries of Name of Institutes Degree Field GPA Graduation Date
graduation
ปรญิ ญาเอก/เทียบเทา Oita University Doctor of Philosophy Medical Science 30/09/2564
Japan

ปริญญาตรี/เทยี บเทา ไทย มหาวทิ ยาลัยมหาสารคาม สตั วแพทยศาสตรบณั ฑติ Veterinary Medicine 3.68 30/04/2560

ประวตั ิการทํางาน Work Experience

ชอ่ื องคก ร/บริษทั ตําแหนงงาน ลักษณะงานทีท่ าํ ระยะเวลาการทํางาน เงินเดือนสุดทาย
Organization / Company Name Position Job Description Starting date - Ending date Last salary (Baht)

วิทยานพิ นธ Thesis

ระดับปรญิ ญา ป พ.ศ. ชื่อเร่อื ง วัตถปุ ระสงคของงานวิจยั หรอื เนอ้ื หาโดยยอ
Degree Year Title Research objectives or summary

ปริญญาเอก 2563 Development of a portable RT-LAMP we establish the CHIKV-specific RT-LAMP primers that target in E1 gene and Dry RT-
system to detect the E1 of CHIKV in a LAMP system which can be applied for the on-site diagnosis
cost-eff

บรรณานกุ รมงานวจิ ยั และงานวิชาการ Research and Academic Work

ช่อื ผูแตง ชือ่ เรื่อง ช่ือวารสาร / สาํ นักพมิ พ ป พ.ศ. จาํ นวนหนา
Author Title Name of Journal or Publisher Year Number of pages
2560
Supawadee Piratae Molecular detection of blood pathogens and their i Asian Pac J Trop Dis 2561 4
2561 10
Yoshiko Gendo Dysbiosis of the Gut Microbiota on the Inflammator Inflammatory intestinal diseases 2562 13
2563 5
Akira Sonoda Oral administration of antibiotics results in feca Genes to Cells 11

Supawadee Piratae Molecular evidence of Ehrlichia canis and Anaplasm Veterinary World

Benjawan Saechue Development of a portable RT-LAMP system to detec Genes to Cells

ทกั ษะ / ความสามารถ Skills / Capabilities

ประเภทคะแนนภาษาอังกฤษ คะแนน วันทที ดสอบ วนั ท่ีหมดอายุ
English Proficiency Test Name Score Test date Expiration date

TOEFL (Internet-based Test : IBT) 60.00 30/06/2564 30/06/2566

ความสามารถทางดา นเทคโนโลยีสารสนเทศและการสื่อสาร :

Information Technology and Communication Skills
Microsoft Office, FlowJo™ Software, ApE- A plasmid Editor, EndNote, Prism software, SPSS

ความรคู วามสามารถพเิ ศษอื่นที่คดิ วา จะเปนประโยชนต อ ตําแหนงท่สี มคั ร :

Other skills that might be beneficial for the position
Problem solving, Adaptability, Handling pressure, High responsibility, Quick learner

ประวัตกิ ารฝกอบรม / สัมนา / ฝก งาน Training / Seminar / Internship Background

หลักสตู ร / วชิ า สถาบนั / บริษทั / องคกร ระยะเวลา
Course og Subject Institutions / Companies / Organizations Training Period

ขาพเจาขอรับรองวาขอ ความ รายละเอียด และเอกสารของขาพเจาทขี่ า พเจา ไดใ หก บั มหาวิทยาลัยในขั้นตอนการสมคั รงานหรือขัน้
ตอนรายงานตวั ในกรณที ่ขี า พเจา เปน ผูไดร บั การคดั เลือก หรอื ทจี่ ะแกไ ขเพ่มิ เตมิ ในอนาคตเปนความจรงิ ทุกประการ ทั้งน้ี ขา พเจาเขาใจดีวา
หากมหาวิทยาลยั รับขา พเจาเขา มาปฏบิ ตั งิ านแลวพบวา มขี อความ หรอื รายละเอียด หรอื เอกสารใดทใี่ หไ วไมเ ปนความจรงิ มหาวิทยาลยั มี
สิทธท์ิ จ่ี ะเลกิ จางขาพเจาไดท ันที โดยไมตองจายเงนิ ชดเชยหรอื คา เสียหายใดๆ ทงั้ สน้ิ

I hereby certify that the text in the application or the details provided or the documents presented are true in all
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SCIENTIFIC SOCIETY
BENJAWAN SAECHUE
§ Poster Presentation:
Veterinarian, DVM o The 42nd annual Meeting of the Molecular Biology Society of Japan, December 3–6,
2019 (Fukuoka, Japan)
PERSONAL DETAILS o The 4th MSUIVC, May 21-22, 2019 (Mahasarakham, Thailand)
o The 41st annual Meeting of the Molecular Biology Society of Japan, November 28-30,
[email protected] 2018 (Yokohama, Japan)
o The 1st Joint Symposium & Workshop Oita University - Universitas Airlangga in
Department of Infectious Medicine "Integrated Medical Life Sciences", June 2-6, 2021 (Zoom webinar, Japan
Disease Control, Faculty of and Indonesia)
Medicine, Oita University,
Oita 879-5593, Japan PUBLICATIONS

(+81) 70-3994-1626 § Saechue B, Kamiyama N, Wang Y, Fukuda C, Watanabe K, Soga Y, Goto M, Dewayani A,
Ariki S, Hirose H, Ozaka S, Sachi N, Hidano S, Faisal K, Chowdhury R, Anik Ashfaq Khan
2nd August 1992 M, Hossain F, Ghosh P, Shirin T, Mondal D, Murakami K, Kobayashi T. “Development of
a portable reverse transcription loop-mediated isothermal amplification system
to detect the E1 region of Chikungunya virus in a cost-effective manner.” Genes
Cells. 2020 Sep;25(9):615-625.
EDUCATION
§ Piratae S, Senawong P, Chalermchat P, Harnarsa W, Saechue B. “Molecular evidence
§ PhD/Medical Science of Ehrlichia canis and Anaplasma platys and the association of infections with
Oita University, JAPAN (MEXT) hematological responses in naturally infected dogs in Kalasin, Thailand.” Vet
2017 – Present World. 2019 Jan;12(1):131-135.
(Expected graduation;
September 2021) § Gendo Y, Matsumoto T, Kamiyama N, Saechue B, Fukuda C, Dewayani A, Hidano S,
Noguchi K, Sonoda A, Ozaki T, Sachi N, Hirose H, Ozaka S, Eshita Y, Mizukami K, Okimoto
§ DVM/Veterinary Medicine T, Kodama M, Yoshimatsu T, Nishida H, Daa T, Yamaoka Y, Murakami K, Kobayashi T.
Mahasarakham University, “Dysbiosis of the Gut Microbiota on the Inflammatory Background due to Lack of
THAILAND Suppressor of Cytokine Signalling-1 in Mice.” Inflamm Intest Dis. 2019 Feb;3(3):145-
2011 – 2016 154.

LANGUAGES ABILITY § Sonoda A, Kamiyama N, Ozaka S, Gendo Y, Ozaki T, Hirose H, Noguchi K, Saechue B,
Sachi N, Sakai K, Mizukami K, Hidano S, Murakami K, Kobayashi T. “Oral
administration of antibiotics results in fecal occult bleeding due to metabolic
§ ENGLISH disorders and defective proliferation of the gut epithelial cell in mice.” Genes Cells.
§ JAPANESE 2018 Dec;23(12):1043-1055.

LICENSE § Noguchi K, Kamiyama N, Hidano S, Gendo Y, Sonoda A, Ozaki T, Hirose H, Sachi N,
Saechue B, Ozaka S, Eshita Y, Mizukami K, Kawano K, Kobayashi T. “Autoimmune
§ Veterinary License sialadenitis is associated with the upregulation of chemokine/chemokine
(01-12301/2560) receptor pairs in T cell-specific TRAF6-deficient mice.” Biochem Biophys Res
Commun. 2018 Sep 26;504(1):245-250.
AWARD
§ Piratae S, Saechue B, Sukumolanan P, Phosri A. “Molecular detection of blood
§ The Best Poster Presentation pathogens and their impacts on levels of packed cell volume in stray dogs from
Award of the 4th MSU Thailand.” Asian Pac. J. Trop. Dis, 2017 April;7(4):233-236.
International Veterinary
Conference (MSUIVC), 2019 LABORATORY SKILLS & EXPERIENCE

§ Establishment of cold chain free-LAMP method for CHIKV and Leishmania detection
§ CRISPR/Cas9 Genome Editing in Mouse using i-GONAD (improved-Genome editing via

Oviductal Nucleic Acids Delivery) method
§ Mouse model of Experimental Autoimmune Encephalomyelitis (EAE)
§ Mouse model of Infectious diseases such as Leishmaniasis, CHIKV, ZIKV, C. rodentium
§ Production of Single-Round Infectious Particles (SRIPs) chimeric Zika virus
§ Flow Cytometric Analysis
§ Other: PCR, RT-PCR, qPCR, qRT-PCR, Western Blot, ELISA, Immunofluorescence Assay

(IFA), Cell line culture, Primary cell culture, Viral culture, Leishmania culture, In vitro
fertilization (IVF) in mouse
§ Teaching assistant of parasitology laboratory practice for medical students (Oita
university, Japan)

Test Taker Score Report

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| |Received: 22 February 2018    Revised: 21 September 2018    Accepted: 9 October 2018 Genes to Cells

DOI: 10.1111/gtc.12649

ORIGINAL ARTICLE

Oral administration of antibiotics results in fecal occult bleeding
due to metabolic disorders and defective proliferation of the gut
epithelial cell in mice

Akira Sonoda1,2   |  Naganori Kamiyama1  |  Sotaro Ozaka1,2  |  Yoshiko Gendo1  | 
Takashi Ozaki1  |  Haruna Hirose1  |  Kaori Noguchi1  |  Benjawan Saechue1  | 
Nozomi Sachi1  |  Kumiko Sakai3  |  Kazuhiro Mizukami2,4  |  Shinya Hidano1  | 
Kazunari Murakami2  |  Takashi Kobayashi1

1Department of Infectious Disease Control, Abstract
Faculty of Medicine, Oita University, Yufu,
Japan Antibiotics sometimes exert adverse effects on the pathogenesis of colitis due
2Department of Gastroenterology, Faculty to the dysbiosis resulting from the disruption of gut homeostasis. However, the
of Medicine, Oita University, Yufu, Japan precise mechanisms underlying colitogenic effects of antibiotic‐induced colitis
3Department of Division of Life Science are largely unknown. Here, we show a novel murine fecal occult bleeding
Research, Faculty of Medicine, Oita model induced by the combinatorial treatment of ampicillin and vancomycin,
University, Yufu, Japan which is accompanied by an enlarged cecum, upregulation of pro‐inflammatory
4Clinical Training Institute for Interns, cytokines IL‐6 and IL‐12, a reduction in Ki‐67‐positive epithelial cell number
Faculty of Medicine, Oita University, Yufu, and an increase in the apoptotic cell number in the colon. Moreover, gas chro-
Japan matography–tandem mass analysis showed that various kinds of metabolites,
including glutamic acid and butyric acid, were significantly decreased in the
Correspondence cecal contents. In addition, abundance of butyric acid producer Clostridiales
Takashi Kobayashi, Oita University, Yufu, was dramatically reduced in the enlarged cecum. Interestingly, supplementa-
Oita, Japan. tion of monosodium glutamate or its precursor glutamine suppressed colonic
Email: [email protected] IL‐6 and IL‐12, protected from cell apoptosis and prevented fecal occult blood
indicating that the reduced level of glutamic acid is a possible mechanism of
Funding information antibiotic‐induced fecal occult bleeding. Our data showed a novel mecha-
Suzuken Memorial Foundation; Japan nism of antibiotic‐induced fecal occult bleeding providing a new insight into
Society for the Promotion of Science, Grant/ the clinical application of glutamic acid for the treatment of antibiotic‐induced
Award Number: 15K08953, 15K19577, colitis.
16K01872, 17H04649, 17H17104,
17K08695, 17K08889, 17K15954 and KEYWORDS
17K16346; GlaxoSmithKline
antibiotic‐induced fecal occult bleeding, epithelial cell proliferation and apoptosis, glutamic acid,
Communicated by: Shigeo Koyasu metabolites, short‐chain fatty acid

Parts of this study were presented at the 40th Annual Meeting of the Molecular Biology Society of Japan, December 6‐9, Kobe, Japan.

|Genes Cells. 2018;23:1043–1055. wileyonlinelibrary.com/journal/gtc © 2018 Molecular Biology Society of Japan and John     1043
Wiley & Sons Australia, Ltd

|1044       Genes to Cells SONODA et al.

1  | INTRODUCTION as induced recuperation from fecal occult blood normalized
cytokine level and protection from epithelial cell apoptosis.
Antibiotics sometimes exert adverse effects on the patho- These results suggest that the reduced level of Glu is a pos-
genesis of colitis. Clostridium difficile‐associated diarrhea sible mechanism of antibiotic‐induced fecal occult bleeding.
(CDAD) and hemorrhagic colitis are known as antibiotic‐in- Our studies show a novel mechanism underlying the colito-
duced colitis. Moreover, antibiotic exposure in childhood is genic effects of antibiotics and provide a new insight into the
a risk factor in inflammatory bowel disease (IBD) and the clinical application of Glu for the treatment of antibiotic‐in-
increasing number of patients being diagnosed with IBD is duced colitis.
becoming a social problem in developed countries (Ng et al.,
2015). These adverse effects of antibiotics on the pathogen- 2  | RESULTS
esis of colitis are highly associated with particular intes-
tinal conditions such as dysbiosis, metabolic disorder and 2.1  |  Combinatorial treatment with
defective epithelial cell growth. For example, mice treated ampicillin and vancomycin inhibits tissue
with a combination of four antibiotics including ampicillin regeneration
(ABPC), vancomycin (VCM), metronidazole (MNZ) and
neomycin (Neo) experienced worsened dextran sulfate so- It has been reported that combinatorial treatment with four
dium (DSS)‐induced colitis associated with the perturbation antibiotics (ABPC, VCM, MNZ and Neo) aggravated DSS‐
of commensal microbiota (Rakoff‐Nahoum, Pglino, Eslami‐ induced colitis in mice (Rakoff‐Nahoum et al., 2004). First,
Varzaneh, Edberg, & Medzhitov, 2004). It has been showed we gavaged these same antibiotics for three consecutive days
that a reduced signal from Toll‐like receptors engaged by and found that all mice tested exhibited occult blood in the
commensal bacteria exacerbates colitis and the administra- feces (n = 5). To determine which of these antibiotics are es-
tion of either lipopolysaccharide (LPS) or lipoteichoic acid sential in inducing fecal occult blood, mice were treated with
can revert the disease phenotype (Rakoff‐Nahoum et al., each antibiotic separately. Mice treated with MNZ or Neo as
2004). The gut microbiota also plays an essential role on the well as untreated control mice tested negative for fecal occult
regulation of metabolism and the immune system (Furusawa blood. Conversely, 40% (2/5) of mice treated with ABPC or
et al., 2013; Matsumoto et al., 2011). For example, VCM VCM tested positive for fecal occult blood. Interestingly, by
treatment in mice shows a strong effect on the gut microbiota combining both antibiotics, ABPC and VCM (A + V) fecal
such as Firmicutes as well as on the intestinal metabolism occult bleeding was induced in all mice (8/8) (Table 1).
of short‐chain fatty acids (SCFAs) such as butyrate (Yap et
al., 2008). Butyrate produced by Clostridia induces differ- Enlargement of the cecum in A + V‐treated mice
entiation of colonic regulatory T cells which is required for (Figure 1a) was also apparent, and the contents of both
the maintenance of gut homeostasis (Furusawa et al., 2013). the cecum and colon were dark‐colored. The weight of the
Moreover, antibiotics alter the expression of genes related to cecum from mice treated with antibiotics was greater than
the cell cycle leading to the reduction of epithelial activity that of control mice. Specifically, the weight of the cecum
in colonic epithelium suggesting that commensal microbiota in mice treated with A + V was approximately three times
drive colonic epithelial proliferation (Reikvam et al., 2011). that of the control mice (Figure 1b). Fecal occult bleeding,
In addition, mice treated with VCM alone exhibited delayed enlarged and dark‐colored cecum were also seen in all mice
recovery from DSS‐induced colitis due to a lack of IL‐22 re- provided A + V in drinking water for 14 days (n = 4).
quired for wound healing of colonic epithelial cells (Zhao et
al., 2016). Despite the breadth of this research, the precise Although histological assessment of the stomach,
mechanisms underlying colitogenic effects of antibiotics are jejunum, cecum and colon did not exhibit any inflammatory
still largely unknown.
T A B L E 1   Number of fecal occult blood positive mice
In the present study, we have showed that a combination
of ABPC and VCM is necessary and sufficient to induce Day 0 Day 1 Day 3
fecal occult bleeding in mice. The combinatorial treatment
of ABPC and VCM (A + V) induced fecal occult blood, CON 0/10 0/10 0/10
enlarged the cecum, upregulated inflammatory cytokines,
reduced the number of Ki‐67‐positive epithelial cells and ABPC 0/5 0/5 2/5
induced apoptosis of epithelial cells in the colon. The me-
tabolomic analysis showed reduced levels of SCFAs and VCM 0/5 2/5 2/5
glutamic acid (Glu). Importantly, supplementation of mono-
sodium glutamate (MSG) or its precursor glutamine (Gln) MNZ 0/5 0/5 0/5
improved the antibiotic‐induced fecal occult bleeding as well
Neo 0/5 0/5 0/5

A + V 0/8 8/8 8/8

Note. ABPC: ampicillin; CON: water alone; MNZ: metronidazole; Neo: neomy-
cin; A + V: ampicillin and vancomycin; VCM: vancomycin.

SONODA et al. Genes to Cells |     1045

F I G U R E 1   Pathogenic aspects and cytokine induction in antibiotic‐induced fecal occult bleeding. (a) Macroscopic findings of the colon
and cecum from mice treated with ampicillin and vancomycin (A + V) for three days. The colon and cecum from control mice (CON) and A + V‐
treated mice (A + V) are shown. (b) Weight of the cecum from mice treated with the indicated antibiotics was measured. (c) mRNA expression of
IL‐6 and IL‐12p40 in the colon measured by real‐time qRT‐PCR method. Each mRNA level was normalized by β‐actin mRNA. (d) Cell‐surface
expression of MHC‐II on CD11b‐positive cells of colonic lamina propria as assessed by flow cytometry. Mean fluorescence intensity (MFI) of
MHC‐II is shown. (e–h) Histological assessment of the colon. Immunohistochemistry for Ki‐67‐positive cells in the colon (e) and enumeration of
Ki‐67‐positive cells per crypt (f) are shown. TUNEL staining of the colon (g) and enumeration of apoptotic cells per field (×400) (h) are shown.
Scale bar: 200 μm. Arrowhead represents apoptotic cell. ABPC: ampicillin; A + V: ampicillin and vancomycin; CON: water alone; Gln: glutamine;
MNZ: metronidazole; MSG: monosodium glutamate; Neo: neomycin; VCM: vancomycin. Statistical significance compared to control groups is
indicated by *: p < 0.05; **: p < 0.01. Data represent mean ± SD (n ≧ 4)

changes (Supporting Information Figure S1), the mRNA ex- pro‐inflammatory cytokines in the colon appears to be an indi-
pression of IL‐6 and IL‐12p40 was slightly increased in the rect effect of the antibiotics, as the antibiotics did not directly
colon of mice treated with A + V (Figure 1c). mRNA ex- induce the cytokines from macrophage cell line RAW264.7
pression of IL‐1β, IL‐10, MIP‐1α, IL‐17A, IFN‐γ, TGF‐β in vitro (data not shown). These results suggest that the asso-
and TNF‐α, however, remained unchanged (Supporting ciation between fecal occult bleeding and immune response is
Information Figure S1B). In the colonic lamina propria, the very limited. We subsequently investigated cell proliferation
proportion of T‐cell subsets, neutrophils and macrophages using immunohistochemical analysis. The mean numbers of
remained unchanged (Supporting Information Figure S1C– Ki67‐positive cells per crypt were significantly reduced in
F), but MHC‐II expression on CD11b‐positive macrophages A + V‐treated mice compared to those in control mice, sug-
was significantly upregulated in A + V‐treated mice (Figure gesting a decreased proliferative capability in epithelial cells
1d, Supporting Information Figure S1G). The induction of (Figure 1e,f). This finding was also seen in the mice treated

|1046       Genes to Cells SONODA et al.

F I G U R E 2   Metagenomic sequence analysis of microbiota in the cecum of mice treated with antibiotics. (a) Changes of the alpha‐diversity
in the cecal contents induced by indicated antibiotics were analyzed using Chao1 indices. Relative abundance of bacterial phylum (b), order (c)
and family (d) compositions in the cecal samples of mice treated with indicated antibiotics was analyzed. The samples are pooled from four to nine
mice. ABPC: ampicillin; A + V: ampicillin and vancomycin; CON: water alone; MNZ: metronidazole; Neo: neomycin; VCM: vancomycin

SONODA et al. Genes to Cells |     1047

with A + V for 24 hr (Supporting Information Figure S1H). also increased Lactobacillales while lowering Clostridiales
Moreover, TUNEL staining showed an increased number as compared with these populations in control mice, at the
of apoptotic colonic epithelial cells in A + V‐treated mice order level (Figure 2c). At the family level, S24‐7, belong-
(Figure 1g,h). These results suggest that the combinatorial ing to phylum Bacteroidetes, was dramatically reduced and
treatment of ABPC and VCM induces fecal occult bleeding Lactobacillaceae was increased by these treatments (Figure
due at least in part to defective tissue regeneration. 2d). These results clearly show that the bacterial composition
of order Lactobacillales, order Clostridiales and family S24‐7
2.2  |  Species richness of the gut microbiota is similarly affected by treatment with ABPC, VCM, or a
was reduced by ABPC and VCM treatment combination of ABPC and VCM. A similar effect was also
seen on the bacterial diversity of mice treated with A + V
We investigated the distribution of microbes in the fecal sam- for 24 hr as seen by the terminal restriction fragment length
ples at the phylum, class, order, family, genus and species polymorphism (T‐RFLP) method (Supporting Information
levels using 16S rRNA gene amplicon sequencing. Alpha‐di- Figure S2).
versity was determined by number of operational taxonomic
units (OTUs), bias‐corrected with Chao1. Chao1 indices 2.3  |  A combined administration of ABPC
showed reduced diversity in mice treated with ABPC, VCM and VCM induces metabolic disorders of
and A + V, which are known as broad‐spectrum antibiotics, carbohydrate, glutamic acid and lipid
over the 3‐day treatment period (Figure 2a). metabolism

The bacterial composition in the cecal contents was an- We observed an enlargement of the cecum in antibiotics‐
alyzed at phylum (Figure 2b), class (data not shown), order treated mice, which resembled that seen in germ‐free (GF)
(Figure 2c), family (Figure 2d) and species level (data not mice. As the enlarged cecum in GF mice is caused by a meta-
shown). A majority of phylum Bacteroidetes were eradicated, bolic disorder, we analyzed the metabolites in the cecal con-
while phylum Firmicutes saw a relative increase in VCM‐ tents using a gas chromatography–tandem mass spectrometer
treated and A + V‐treated mice (Figure 2b). A + V treatments

F I G U R E 3   Metabolites in the cecal
samples from mice treated with antibiotics
were analyzed by GC/MS/MS‐TQ8040. (a)
Changes of short‐chain fatty acids (SCFAs)
were analyzed with BPX‐5 column on
TQ8040. Relative intensity of indicated
SCFAs is graphed. n ≧ 4. (b) Changes
of metabolites were analyzed with DB‐5
column on TQ8040. Relative intensity of
indicated metabolites is graphed. n ≧ 5.
ABPC: ampicillin; A + V: ampicillin and
vancomycin; CON: water alone; meto:
methoximation; MNZ: metronidazole;
N.D.: not detected; Neo: neomycin; TMS:
trimethylsilylation; VCM: vancomycin.
Statistical significance compared to
control groups is indicated by *p < 0.05,
**p < 0.01, Data represent mean ± SD

|1048       Genes to Cells SONODA et al.

F I G U R E 4   The effect of butyrate on
the production of cytokine and antimicrobial
peptide in vitro and in vivo. (a) Mouse
macrophage cell line RAW267.4 was
left untreated (CON) or stimulated with
lipopolysaccharide (LPS: 3 ng/ml), with or
without sodium butyrate (SB: 1 mM) for
24 hr. The mRNA expression fold changes
of indicated cytokines are shown. n = 5.
Each mRNA level was normalized by β‐
actin mRNA. (b) Mouse colon cell line,
CMT93, was stimulated with SB (1 mM)
for 24 hr. The mRNA expression fold
changes of indicated antimicrobial peptides
are shown. mBD3: mouse beta defensin
3; SLPI: secretory leukocyte protease
inhibitor. n = 5. (c) The mRNA expressions
of indicated cytokines in the colon of mice
treated with Clostridium Butyricum (CB)
or SB were measured by real‐time qRT‐
PCR method. n ≧ 4. *: p < 0.05. N.S.: not
significant. Data represent mean ± SD. Each
mRNA level was normalized by β‐actin
mRNA

(GC/MS/MS) with BPX‐5 column that allows the measure- 2.4  |  Butyric acid prevents a macrophage
ment of short‐chain fatty acids (SCFAs). As shown in Figure cell line from inducting cytokines and induces
3a and Supporting Information Figure S3, butyric acid, acetic antimicrobial peptides in a colon carcinoma
acid, propionic acid and valeric acid were significantly re- cell line
duced in ABPC‐treated mice and almost completely elimi-
nated in VCM‐treated and A + V‐treated mice during the To show the pathophysiological relevance of metabolites af-
three days of treatment. On the contrary, formic acid experi- fected by antibiotics treatment, first we examined the effects
enced a significant increase in these mice. of butyrate on monocytes. It has been reported that butyrate
decreases the expression of cytokines in peripheral blood
Next, we analyzed other metabolites in the cecal contents mononuclear cells from patients with Crohn’s disease (CD)
using a GC/MS/MS with DB‐5 column. Twenty‐nine metab- in response to LPS (Segain et al., 2000). Consistent with pre-
olites were significantly changed with antibiotics treatment vious reports, LPS‐induced mRNA expression of MIP‐1α,
(Supporting Information Table S1). Metabolites which in- TNFα and IL‐1β in macrophage cell line RAW264.7 was sig-
creased in A + V‐treated mice were mainly lactic acid and nificantly suppressed by sodium butyrate (SB) (Figure 4a).
carbohydrates (ribonic acid, arabitol, sorbose and 2‐deoxy‐
glucose). Contrarily, aromatic substances (caproic acid, ca- As butyrate induces the expression of an antimicrobial
daverine, octanoic acid and 5‐aminovaleric acid), glutamic peptide, cathelicidin, in human colon carcinoma cell line
acid (Glu) and its metabolites (nicotinic acid, alanine and HT29 cells (Steinmann, Agerberth, & Gudmundsson, 2009),
uracil) were significantly reduced in mice treated with A + V we further investigated the effect of butyrate treatment on the
within the 3‐day treatment period (Figure 3b, Supporting colon epithelial cells. A mouse colon carcinoma cell line,
Information Figure S3B, Table S1). These results suggest CMT93, was stimulated with 10 mM SB for 24 h. As shown
that antibiotic(s) treatment disturbs the metabolism of Glu, in Figure 4b, SB induced mRNA expression of secretory leu-
carbohydrates, aromatic substances and SCFAs in the cecum kocyte proteinase inhibitor (SLPI) and lactoferrin, which are
of mice. known not only as antimicrobial peptides, but also as potent

SONODA et al. Genes to Cells |     1049

F I G U R E 5   Glutamate and glutamine treatment for the antibiotic‐induced fecal occult bleeding. (a) Mice were given 3 g/dl of monosodium
glutamate (MSG) or glutamine (Gln) in drinking water (Wat) or water alone for 6 days. The mice were orally gavaged antibiotics (1.0 mg
ampicillin: A and 0.5 mg vancomycin: V) for 3 days. Then, the mice were examined for occult blood in the feces. (b) The graph shows the
percentage of fecal occult blood positive mice treated with the indicated regimen. (c) Macroscopic findings of the cecum from mice treated with
the indicated regimen. (d) The mRNA expressions of indicated cytokines in the colon as measured by real‐time qRT‐PCR method. *p < 0.05,
**p < 0.01, Data represent mean ± SD. n ≧ 8. Each mRNA level was normalized by β‐actin mRNA

anti‐inflammatory agents in the colon (Ho, Charalabos, & that although the reduced number of Ki‐67‐positive cells in
Koon, 2013). However, mRNA expression of SLPI and lacto- A + V‐treated mice remained unchanged (Figure 1e,f) by
ferrin in A + V‐treated mice was unchanged by SB treatment treatment with MSG or Gln, apoptotic cells were signifi-
(data not shown). Mouse β‐defensin 3 (mBD3), which is the cantly reduced (Figure 1g,h).
most abundant antimicrobial peptide in the colon, was un-
changed (Figure 4b). These results suggest that the retrieval of Glu and Gln in
A + V‐treated mice in which Glu was reduced in the cecum
Oral administration of SB failed to improve fecal occult can improve antibiotic‐induced fecal occult bleeding. The re-
bleeding in vivo. The expression levels of IL‐6 and IL‐12p40 duced level of Glu in the cecum is a possible cause of epithe-
remained unchanged (Figure 4c). Supplementation of butyric lial cell death leading to fecal occult bleeding.
acid producer, Clostridium Butyricum, to A + V‐treated mice
showed a limited effect including the slightly reduced expres- 3  | DISCUSSION
sion of IL‐6 and IL‐12p40 (Figure 4c).
We have showed that a combination of ABPC and VCM
2.5  |  Oral administration of monosodium is necessary and sufficient to induce fecal occult bleeding.
glutamate and glutamine prevents antibiotic‐ Some of the VCM‐treated mice tested positive for fecal oc-
induced fecal occult bleeding cult blood and altered cytokine expression, metabolism and
microbial composition in a similar manner to those of the
Monosodium glutamate (MSG) or its precursor glutamine mice treated with A + V. In the present study, we focused
(Gln) was administered orally to A + V‐treated mice (Figure on A + V‐treated mice because the disease phenotype was
5a). 37.5% of MSG and 25% of Gln‐treated mice tested nega- consistently observed within 24 hr of the initial treatment.
tive for fecal occult blood (Figure 5b). Consistent with these Moreover, all mice provided A + V ad libitum in drinking
results, the cecal contents were no longer dark‐colored al- water for 14 days also exhibited positive for fecal occult
though the ceca remained enlarged (Figure 5c). bleeding. These phenotypes could be induced by A + V
regardless of administrating pass. We observed increased
Gln and MSG reduced the expression of colonic IL‐6
and IL‐12p40, respectively. (Figure 5d). We also found

|1050       Genes to Cells SONODA et al.

levels of IL‐6, Il‐12p40 and MHC‐II in A + V‐treated mice. supplementation with sodium butyrate failed to improve
However, as the proportions of T‐cell subsets, neutrophils A + V‐induced fecal occult bleeding (Figure 4c). Although
and macrophages in the colonic lamina propria were not af- butyrate producer Clostridium Butyricum was found to
fected by A + V treatment, the involvement of immune cells slightly suppress the expression of IL‐6 and IL‐12p40 in the
is suspected to be very limited to the fecal occult bleeding. colon (Figure 4c), the precise mechanism of which remains
unclear.
An enlarged cecum was observed in the mice treated
with antibiotics (Figure 1a,b), which resembles that of GF The composition of gut microbiota and the metabolic
mice (Furusawa et al., 2013). The enlarged cecum in GF profile were similar among mice treated with ABPC, VCM
mice is caused by metabolic disorder due to the absence of or A + V (Figure 3). Lactic acid and its producer, the order
Clostridiales producing SCFAs. We also observed a signif- Lactobacillales, were found to be increased in A + V‐treated
icant reduction of SCFAs in A + V‐treated mice consistent mice (Figure 6, left). The accumulation of lactate in the large
with that found in a previous report (Yap et al., 2008). Among intestine lowers the luminal pH (Bustos et al., 1994), leading
the SCFAs, butyric acid is the most important nutrient for the to diarrhea (Saunders & Sillery, 1982). In fact, it has been re-
colon epithelium (Kripke, Fox, Berman, Settle, & Rombeau, ported that the level of lactic acid was higher in patients with
1989; Sakata, 1987) and the induction of antimicrobial pep- UC and antibiotic‐induced colitis (Hashizume, Tsukahara, &
tide LL‐37 (Canani et al., 2011). Butyric acid has also been Yamada, 2003; Vernia et al., 1988). Moreover, some of the
reported to improve DSS‐induced colitis in mice (Simeoli et bacteria belonging to order Clostridiales convert lactate into
al., 2016) and ulcerative colitis (UC) (Canani et al., 2011). butyrate (Hashizume et al., 2003).

In the present study, SCFAs and its producer order In our experiments, family S24–7, which is predicted
Clostridiales experienced a marked decrease with A + V to metabolize carbohydrates, was remarkably reduced in
treatment. Furthermore, aromatic substances including A + V‐treated mice. S24‐7 is known to inhabit the gut of
caproic acid, cadaverine, valeric acid and octanoic acid were homeothermic animals, especially herbivores (Ormerod et
also decreased. These findings suggest that bacterial fermen- al., 2016). It has been reported that an increase in S24‐7 oc-
tation of SCFAs is disturbed by antibiotics treatment. In our curs during the healing stage of enteritis (Rooks et al., 2014),
experiments, butyrate suppressed the induction of pro‐in- and induces T‐cell‐dependent IgA production (Bunker et al.,
flammatory cytokines IL‐1β, MIP‐1α and TNFα in a macro- 2015). The physiological implication of S24‐7 in the fecal
phage cell line, and induced antimicrobial peptides, SLPI and occult bleeding remains uncertain, as they are uncultur-
lactoferrin, in a colon epithelial cell line. To our knowledge, able. Phylum Proteobacteria was increased in A + V‐treated
this is the first report of butyrate inducing SLPI and lactofer- mice. This phylum includes Acinetobacter which induces
rin from colon epithelial cells. However, IL‐1β, MIP‐1α and opportunistic infection, as well as Klebsiella, Proteus and
TNFα did not experience an increase in A + V‐treated mice. Escherichia which are all related to intestinal inflammation,
Thus, the reduction of butyric acid in A + V‐treated mice is suggesting a colitogenic environment. For example, inocula-
not likely to be the cause of the fecal occult bleeding. Indeed, tion of Klebsiella isolated from CD patients could induce Th1

F I G U R E 6   A model for the
mechanism underlying the development of
the antibiotic‐induced fecal occult bleeding.
Possible mechanisms of the antibiotic‐
induced fecal occult bleeding are shown.
See “Discussion” section

SONODA et al. Genes to Cells |     1051

cells in the colon of recipient mice (Atarashi et al., 2017). most abundant amino acid in an elemental diet and is tra-
In the present study, these colitogenic bacteria were not de- ditionally used for treatment of CD in Japan (Takagi et al.,
tected at species level. The abundance of Acinetobacter rhi- 2006); however, it is not recommended by European guide-
zosphaerae, belonging to phylum Proteobacteria and often lines (Gomollón et al., 2017) because of insufficient evidence
shown to exhibit plant growth‐promoting traits when isolated and an unknown mechanism. Our data suggest that dysreg-
from the rhizosphere, was increased by A + V treatment (data ulation of Glu metabolism induced by antibiotics is highly
not shown) (Gulati, Vyas, Rahi, & Kasana, 2009). Warn et al associated with the development of IBD and provide a new
reported that VCM increased the abundance of Acinetobacter insight into the clinical application of Glu for the treatment
rhizosphaerae (Warn et al., 2016); however, the pathophys- of antibiotic‐induced colitis. Further investigation is required
iological relevance is still unclear. Further investigation is to show the precise mechanism for how antibiotics disturb
needed to show the physiological role of Acinetobacter rhizo- Glu metabolism and its associated commensal bacteria in
sphaerae in the maintenance of gut homeostasis. bowel diseases. From these results, the precise mechanisms
of fecal occult bleeding induced by antibiotics could not be
It is worthy to note here that the levels of Glu metabo- entirely confirmed and we cannot exclude the possibility of
lites including nicotinic acid and alanine were reduced in the harmful effect of antibiotics on the epithelial cells. For exam-
cecal contents by treatment with A + V. Both Glu and Gln ple, A + V treatment may induce defective tissue regenera-
are known as the critical energy sources of the gut epithelial tion leading to the epithelial damage and bleeding, which in
cells (Blachier, Boutry, Bos, & Tomé, 2009; Burrin, 2002). turn results in dysbiosis, and ultimately induces colitis at the
These are provided from luminal and arterial sources. Our late phase. Alternatively, we also cannot exclude the involve-
data show that the Glu level was remarkably reduced in the ment of gut microbiota and microbial metabolism because
cecal contents of A + V‐treated mice suggesting that the mu- metabolites and microbiota were affected 24 hr following
cosal Glu metabolism derived from dietary intake was im- A + V treatment (Supporting Information Figures S2 and
paired. However, the commensal bacteria which produce Glu S3). Importantly, supplementation with MSG did improve
in the gut microenvironment are still unidentified (Figure 6, the outcome of fecal occult bleeding with protection from
right). Glu is converted to Gln in the colonic cells, as well as apoptosis. These data may at least suggest that the effects of
butyrate and acetate in the microflora, which in turn is used antibiotics on the fecal occult bleeding are associated with
by the villi to maintain their integrity (Blachier et al., 2009). defective tissue regeneration.
Gln has been shown to exert pluripotent actions, including
stimulation of cell proliferation and suppression of apoptosis 4  | EXPERIMENTAL
(Kandil et al., 1995; Papaconstantinou et al., 1998). In our PROCEDURES
data, combinatorial treatment with ampicillin and vancomy-
cin reduced the level of Glu and the number of Ki‐67‐positive 4.1  | Mice
epithelial cells and increased the number of apoptotic colonic
epithelial cells concomitantly. Interestingly, MSG and Gln Six‐ to eight‐week‐old male C57BL/6J mice were purchased
treatment provided protection from epithelial cell apoptosis. from CLEA Japan (Tokyo, Japan). Mice were given a routine
These results suggest that the combinatorial treatment with chow diet (LabDiet 500N, PMI Nutrition International, INC.
ABPC and VCM induces fecal occult bleeding due at least in MI) and autoclaved water under specific pathogen‐free con-
part to defective tissue regeneration caused by an imbalance ditions. This animal experiment was approved by and carried
between cell proliferation and cell death of intestinal epithe- out according to the guidelines of the Oita University Animal
lial cells. Ethics Committee.

It has been reported that Glu prevents nonsteroidal anti‐ 4.2  |  Antibiotics treatment and
inflammatory drug‐induced enteritis (Amagase et al., 2012). examination of occult blood in the feces
Moreover, Gln supplementation attenuates experimental
colitis due to the inhibition of inflammatory signaling in- Mice were gavaged with 1 mg/body ABPC (Nacalai Tesque,
cluding NF‐κB and STAT, or the inhibition of T‐cell migra- Inc, Kyoto, Japan), 0.5 mg/body VCM (Nacalai Tesque,
tion (Simeoli et al., 2016; Steinmann et al., 2009; Takagi et Inc), 1 mg/body MNZ (Sigma‐Aldrich Co. Ltd, St. Louis,
al., 2006; Vernia et al., 1988). In our experiments, both Glu MO, USA), 1 mg/body Neo (Nacalai Tesque, Inc) or a com-
and Gln partially suppressed the A + V‐induced fecal oc- bination of 1 mg/body ABPC and 0.5 mg/body VCM using
cult bleeding, although the suppression mechanism remains a stomach sonde (Fuchigami, Kyoto, Japan) every 24 hr for
unknown. three consecutive days. Control mice were given 300µl of
distilled water at the same interval. Examination of occult
In a clinical setting, Gln level is decreased in the colonic blood in the feces was carried out using Hemoccult (Beckman
mucosa in patients with active UC (Shiomi et al., 2011), and
the supplementation of Gln improves the disease outcome
(Cöeffier, Marion‐Letellier, & Déchelotte, 2010). Glu is the

|1052       Genes to Cells SONODA et al.

Coulter, Inc, Brea, CA, USA) on day 0 (before administration GCASCAG‐3′,andreverseprimer5′‐CAAGCAGAAGACGG
of antibiotics), day 1 (24 hr after the first administration) and CATACGAGATZZZZZZZZGTGA
day 3 (24 hr after the last administration). Some mice were CTGGAGTTCAGACGTGTGCTCTTCCGATCTGACTA
given antibiotics with drinking water (ampicillin 1 g/L and CNVGGGTATCTAATCC‐3′, the underlined sequences rep-
vancomycin 0.5 g/L) for 14 days. resent the PCR primer region (Pro341F and Pro805R). The
libraries were sequenced on the MiSeq platform using MiSeq
4.3  |  Histological analysis Reagent Kit v3 with 2 × 301 cycles. The adapter sequences
and low‐quality regions were trimmed by Cutadapt 1.1 and
Mice were euthanized by cervical dislocation 24 hr after the Trimmomatic 0.32, respectively. Paired‐end sequence reads
last administration of antibiotics at which time the weight of were assembled using the script fastq‐join‐1.1.2‐537. Further
the cecum with their contents was measured. Tissue samples data processing was carried out using the open‐source soft-
were fixed with 10% formalin, embedded in paraffin, sec- ware pipeline “Quantitative Insights into Microbial Ecology”
tioned at 3 μm and stained with hematoxylin and eosin. For (QIIME) version 1.8.0. Reads were trimmed of primers, and
detection of Ki‐67, immunohistochemistry was carried out sequence quality control was carried out using QIIME’s script
using rat monoclonal anti‐mouse Ki‐67 antibody diluted 1:50 (sequences length 200–1,000 nucleotides; minimum average
(Agilent Technologies, CA, USA). Samples were blocked quality score 25; maximum length of homopolymer runs 6).
with Protein Block Serum‐Free (Agilent Technologies) to
reduce nonspecific binding at room temperature for 10 min Operational taxonomic units (OTUs) were formed at 97%
and then incubated at 4°C overnight with anti‐mouse Ki‐67 similarity using UCLUST (de novo OTU picking). The phy-
antibody in Antibody Diluent (Agilent Technologies). The logenetic assignment of representative sequences from each
sections were incubated at room temperature for 30 min with OTU was carried out with a 16S reference dataset obtained
N‐Histofine Simple Stain Mouse MAX‐PO (RAT) (Nichirei, from Greengenes version 13_8, and chimeric sequences were
Tokyo, Japan). The mean numbers of Ki67‐positive cells removed using ChimeraSlayer. Summary of taxonomic as-
were calculated by counting positive cells in 10 crypts in each signments was plotted, and alpha diversities were calculated
transverse section. For detection of apoptotic cells, TUNEL by QIIME.
staining was carried out using TUNEL Apoptosis Detection
Kit (GenScript, NJ, USA), according to the manufacturer’s 16S rRNA T‐RFLP was carried out by
instructions. The mean numbers of apoptotic cells were cal- TechnoSuruga Laboratory Co, Ltd. (Shizuoka, Japan) as de-
culated by counting positive cells in three fields (400 × ob- scribed previously (Nagashima, Hisada, Sato, & Mochizuki,
jective) within each section. Samples were observed under a 2003). Fecal DNA was amplified by PCR using forward primer
microscope (BX43, Olympus, Tokyo, Japan) mounted with 516f (5′‐TGCCAGCAGCCGCGGTA‐3′) and reverse primer
DP27 camera system in a blinded fashion. 1510r (5′‐GGTTACCTTGTTACGACTT‐3′), digested with
BslI and analyzed by ABI PRISM 3,130 x1 Genetic Analyzer
4.4  |  16S rRNA metagenomic analysis and System (Thermo Fisher Scientific, Waltham, MA, USA). The
16S rRNA terminal restriction fragment length phylogenetic assignment of OTU was carried out according
polymorphism (T‐RFLP) of cecal microbiota to reference Human Fecal Microbiota T‐RFLP Profiling
(https://www.tecsrg.co.jp/t-rflp/t_rflp_hito_OTU.html).
16S rRNA metagenomic analysis was carried out as de-
scribed previously (Hisada, Endoh, & Kuriki, 2015). 4.5  |  Gas chromatography–tandem mass
Isolation of DNA, construction of libraries, deep sequenc- spectrometer analysis for metabolites in the
ing and read assembly of the libraries were carried out by cecal contents
Hokkaido System Science (Sapporo, Hokkaido, Japan). Fecal
samples from the cecum of four to nine mice were pooled. Metabolites in the cecal contents were determined by a gas
Fecal DNA was extracted using an automatic nucleic acid chromatography–tandem mass spectrometer (GC/MS/MS),
extraction system (Magtration System 12GC, Precision GC/MS‐TQ8040 (Shimazu, Kyoto, Japan). A BPX‐5 col-
System Science, Japan). The 16S rRNA gene amplicon li- umn (30 m × 0.25 mm i.d; film thickness 1.00 µm, Trajan
braries were prepared following the “16S Metagenomic Scientific and Medical, Vic., Australia) was used for SCFAs,
Sequencing Library Preparation Protocol” (Illumina, Inc., and a DB‐5 column (30 m × 0.25 mm i.d; film thickness
San Diego, CA, USA) using the Nextera XT index kit with 1.00 µm, J&W Scientific Inc, Folsom, CA, USA) was used
the following primers (for the V3–V4 region of 16S rDNA for other metabolites.
of prokaryotes): forward primer 5′‐AATGATACGGCG
ACCACCGAGATCTACACXXXXXXXXACACTCTT Short‐chain fatty acids in the cecal contents were extracted
TCCCTACACGACGCTCTTCCGATCTCCTACGGGNB with methanol. 180 µl of supernatant was mixed with 20 μl of
100 mM n‐octylamine‐DMT‐MM and then applied on a GC/
MS/MS. For other metabolites, cecal samples extracted with
methanol were added to 1.0 mg/ml isopropylmalic acid as an

SONODA et al. Genes to Cells |     1053

internal standard with distilled water and chloroform. 20 μl Eagle’s medium (DMEM; Thermo Fisher Scientific) with
of supernatant was dried up and then mixed with 120 μl of 10% FCS, at 37°C in 5% CO2. RAW 267.4 cells were stimu-
20 mg/ml methoxyamine hydrochloride in pyridine to oximate. lated with LPS (3 ng/ml) for 24 hr in the presence or absence
Derivatization was carried out using 60 µl of N‐methyl‐N‐ of 10 mM sodium butyrate or antibiotics. CMT93 cells were
trimethylsilyltrifluoroacetamide and then applied on a GC/ treated with 10 mM SB for 24 hr. Cells were then harvested
MS/MS. The analysis conditions are shown in Table 2. Peaks for the measurement of cytokines by real‐time quantitative
were detected using the GCMSsolution software (Shimazu), reverse transcription–polymerase chain reaction (qRT‐PCR)
and the alkane series mixture (C9 to C33) was used to stan- analysis.
dardize retention time. Metabolites were detected by the Smart
Metabolites Database (Shimazu) which registered 12 spec- 4.8  |  RNA extraction and real‐time
trums for BPX‐5 column and 475 spectrums for DB‐5 column. qRT‐PCR

4.6  |  Glutamate, glutamine, sodium Tissue samples from the colon or cell lines were homog-
butyrate and Clostridium Butyricum treatment enized in TRI Reagent (Cosmo BIO Co. Ltd, Tokyo,
for the antibiotic‐induced fecal occult bleeding Japan), and then total RNA was extracted according to
the manufacturer’s instructions. cDNA synthesis was car-
Monosodium L‐glutamate monohydrate (MSG: Wako Pure ried out using a Verso cDNA Synthesis Kit (Thermo
Chemical Industries, Ltd, Kyoto, Japan), L‐glutamine (Gln: Fisher Scientific Inc). qRT‐PCR was carried out using
Sigma‐Aldrich Co. Ltd), sodium butyrate (SB; Wako Pure a KAPA SYBR FAST qPCR Kit (Kapa Biosystems,
Chemical Industries, Ltd) or probiotics including Clostridium Wilmington, MA, USA) with a fluorescence temperature
Butyricum (CB, Miyarisan, Tokyo, Japan) were orally ad- cycler (LightCycler 96, Roche Diagnostics, Indianapolis,
ministered to the antibiotic‐induced fecal occult bleeding in IN, USA). The sequences of primers are listed as follows:
mice. A + V‐treated mice were given 3 g/dl of MSG or 3 g/ β‐actin F: CTTCCTCCCTGGAGAAGAGCTATGAGC, R:
dl Gln or 2 g/dl SB three days before the antibiotics treat- GCCTAGAAGCACTTGCGGTGCACG; IL‐1β F: GAG
ment. 6 mg of CB was orally administered by stomach sonde TGTGGATCCCAAGCAAT, R: TACCAGTTGGGGAAC
together with antibiotics for three days. TCTGC; IL‐6 F: CCGGAGAGGAGACTTCACAG, R: CA
GAATTGCCATTGCACAAC; IL‐10 F: GGTTGCCA
4.7  |  Cell lines, cell culture and treatments AGCCTTATCGGA, R: AAATCGATGACAGCGCCTC;
IL‐12p40 F: CCTGAAGTGTGAAGCACCAA, R: TCA
Mouse monocyte cell line RAW267.4 (ATCC, Manassas, GGGGAACTGCTACTGCT; IL‐17A F: GGCCCTCAG
VA, USA) and mouse colon carcinoma cell line CMT93 ACTACCTCAACC, R: TGAGCTTCCCAGATCACAGAG;
(ECACC, Salisbury, UK) were grown in Dulbecco’s modified TGFβ F: CATCCATGACATGAACCGGC, R: CTTCT
CTGTGGAGCTGAAGCA; IFNγ F: ATGAACGCTA
T A B L E 2   Analysis conditions of gas chromatography–tandem CACACTGCATC, R: CCATCCTTTTGCCAGTTCCTC;
mass spectrometry TNF‐α F: CCACCACGCTCTTCTGTCTA, R: TCCTCCAC
TTGGTGGTTTGT; MIP‐1α F: TGCCCTTGCTGTTCTTC
Column BPX−5 DB−5 TCT, R: GATGAATTGGCGTGGAATCT; secretory leuko-
cyte inhibitor (SLPI) F: GGCCTTTTACCTTTCACGGTG,
Glass insert Split mode (split Splitless mode R: TACGGCATTGTGGCTTCTCAA; mouse β difen-
ratio 1:30) sin3 (mBD3) F: CTTTGCATTTCTCCTGGTGC, R: GCC
Injection volume 1.0 µl 100°C (4 min) TCCTTTCCTCAAACT; lactoferrin F: TCAAGAAAT
Column temperature 60°C (2 min) →10°C/min CCTCCACCCGC, R: ACACGAGCTACACAGGTTGG.
→15°C/min →320°C (11 min) Amplification conditions were as follows: 45 cycles of
Interface →330°C (3 min) 95°C (10 s), 60°C (20 s) and 72 C (1 s). All data were ana-
temperature 280°C lyzed by LightCycler software (Roche Diagnostics, Basel,
Carrier gas Switzerland) and normalized to the β‐actin expression. The
Electron ionization Helium, constant flow rate: 39.0 cm/min fold difference relative to the β‐actin was shown.
voltage 70 eV
Ion source
temperature 200°C 4.9  |  Isolation of intestinal lymphocytes and
Mass range myeloid cells
Measurement mode 20–200 m/z, 0.3 s pre‐scan velocity
Multiple reaction monitoring Colons were opened longitudinally, placed in HBSS with
5 mM EDTA and incubated at 37°C for 15 min in a shaking

|1054       Genes to Cells SONODA et al.

water bath. After washing in PBS, muscle layers were re- acid medicine: Prophylactic and healing promoting effect of mono-
moved with tweezers. The tissues were then cut into small sodium glutamate against NSAID‐induced enteropathy. Journal
pieces and incubated with RPMI1640 containing 4% FBS, of Pharmacological Sciences, 118(2), 131–137. https://doi.
1 mg/ml collagenase type II, 0.5 mg/ml dispase and 25 μg/ org/10.1254/jphs.11R03FM
ml DNase I for 35 min at 37 °C in a shaking water bath. The Atarashi, K., Suda, W., Luo, C., Kawaguchi, T., Motoo, I., Narushima,
digested tissues were filtered through a 40‐μm cell strainer. S., … Honda, K. (2017). Ectopic colonization of oral bacteria in
the intestine drives TH1 cell induction and inflammation. Science,
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Blachier, F., Boutry, C., Bos, C., & Tomé, D. (2009). Metabolism and
The following antibodies were used: anti‐CD3: PE (Thermo functions of L ‐glutamate in the epithelial cells of the small and
Fisher Scientific), anti‐CD4: FITC, anti‐CD8: PerCP Cy5.5, large intestines. American Journal of Clinical Nutrition, 90(3), 814–
anti‐Foxp3: APC, anti‐CD11b: anti‐F4/80, anti‐MHC classII: 821. https://doi.org/10.3945/ajcn.2009.27462S
Alexa Fluor 700 (BioLegend, CA, USA) and zombie red Bunker, J. J., Flynn, T. M., Koval, J. C., Shaw, D. G., Meisel, M.,
(BioLegend) after blockade of Fc receptors. The intracel- McDonald, B. D., … Bendelac, A. (2015). Innate and adaptive
lular expression of Foxp3 in CD4+ T cells was analyzed humoral responses coat distinct commensal bacteria with immu-
using a Foxp3/Transcription Factor Staining Buffer Set noglobulin A. Immunity, 43(3), 541–553. https://doi.org/10.1016/j.
(Thermo Fisher Scientific) according to the manufacturer’s immuni.2015.08.007
instructions. Data were acquired using a FACSVerse or a Burrin, D. G. G. (2002). Gastrointestinal protein and amino acid metabo-
LSRFortessa (BD Biosciences, NJ, USA) and analyzed using lism in growing animals. Biology of the Intestine in Growing Animals,
FlowJo software (BD Biosciences). 1, 695–725. https://doi.org/10.1016/S1877-1823(09)70140-7
Bustos, D., Pons, S., Pernas, J. C., Gonzalez, H., Caldarini, M. I.,
4.11  |  Statistical analysis Ogawa, K., & Paula, J. A. D. E. (1994). Fecal lactate and short
bowel syndrome. Digestive Diseases and Sciences, 39(11), 2315–
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Differences between two groups were analyzed by Student’s t Canani, R. B., Costanzo, M. D., Leone, L., Pedata, M., Meli, R.,
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more groups using GraphPad Prism 6.0 (GraphPad Software ate in intestinal and extraintestinal diseases. World Journal of
Inc., San Diego, CA, USA). p values <0.05 were considered Gastroenterology, 17(12), 1519–1528. https://doi.org/10.3748/wjg.
to be significant. v17.i12.1519
Cöeffier, M., Marion‐Letellier, R., & Déchelotte, P. (2010). Potential
ACKNOWLEDGMENTS for amino acids supplementation during inflammatory bowel dis-
eases. Inflammatory Bowel Diseases, 16, 518–524. https://doi.
The authors thank Ms. Chiharu Aoki for her secretarial as- org/10.1002/ibd.21017
sistance; Ms. Mami Koya, Ms. Yoko Kudo and Ms. Kanako Furusawa, Y., Obata, Y., Fukuda, S., Endo, T. A., Nakato, G., Takahashi,
Ito for providing technical assistance; and the members D., … Ohno, H. (2013). Commensal microbe‐derived butyrate in-
of Dr. Kobayashi’s laboratory for valuable discussions. duces the differentiation of colonic regulatory T cells. Nature,
This work was supported by Aid from Japan Society for 504(7480), 446–450. https://doi.org/10.1038/nature12721
the Promotion of Science [17K15954 to A.S.], [15K08953 Gomollón, F., Dignass, A., Annese, V., Tilg, H., Assche, G. V., Lindsay,
to K. Mizukami], [16K01872 to K.S.], [17K08695 to J. O., … Gionchetti, P. (2017). ECCO guideline/consensus paper
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[15K19577 to T. O.], [17H17104 to N.K.], [17K15680 agement of Crohn’s disease 2016: Part 1: Diagnosis and medical
to S.H.], [17K16346 to H.H.]; the Suzuken Memorial management. Journal of Crohn's and Colitis, 11(1), 3–25. https://
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Grant to S.H. Gulati, A., Vyas, P., Rahi, P., & Kasana, R. C. (2009). Plant growth‐
promoting and rhizosphere‐competent acinetobacter rhizos-
ORCID phaerae strain BIHB 723 from the cold deserts of the himalayas.
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| |Received: 27 December 2019    Revised: 1 June 2020    Accepted: 10 June 2020 Genes to Cells

DOI: 10.1111/gtc.12797

ORIGINAL ARTICLE

Development of a portable reverse transcription loop-mediated
isothermal amplification system to detect the E1 region of
Chikungunya virus in a cost-effective manner

Benjawan Saechue1  | Naganori Kamiyama1  | Yinan Wang1  | Chiaki Fukuda1  |
Kei Watanabe1  | Yasuhiro Soga1  | Mizuki Goto1  | Astri Dewayani1  | Shimpei Ariki1  |
Haruna Hirose1  | Sotaro Ozaka1  | Nozomi Sachi1  | Shinya Hidano1  | Khaledul Faisal2  |
Rajashree Chowdhury2  | Md. Anik Ashfaq Khan2  | Faria Hossain2  | Prakash Ghosh2  |
Tahmina Shirin3  | Dinesh Mondal2  | Kazunari Murakami4  | Takashi Kobayashi1

1Department of Infectious Disease Control, Abstract
Faculty of Medicine, Oita University, Oita,
Japan Chikungunya fever is a mosquito-borne disease cause of persistent arthralgia. The
2Nutrition and Clinical Services Division, current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse
International Centre for Diarrhoeal Disease transcription polymerase chain reaction assay. Reverse transcription loop-mediated
Research, Bangladesh (icddr, b), Dhaka, isothermal amplification (RT-LAMP) is a rapid and simple tool used for DNA-based
Bangladesh diagnosis of a variety of infectious diseases. In this study, we established an RT-
3Department of Virology, Institute of LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene
Epidemiology Disease Control and that could also detect CHIKV at a concentration of 8 PFU without incorrectly detect-
Research (IEDCR), Dhaka, Bangladesh ing other mosquito-borne viruses. The system also amplified the E1 genome in the
4Department of Gastroenterology, Faculty serum of CHIKV-infected mice with high sensitivity and specificity. Moreover, we
of Medicine, Oita University, Oita, Japan established a dry RT-LAMP system that can be transported without a cold chain,
which detected the virus genome in CHIKV-infected patient samples with high ac-
Correspondence curacy. Thus, the dry RT-LAMP system has great potential to be applied as a novel
CHIKV screening kit in endemic areas.
Takashi Kobayashi, Oita University, 1-1
Idaigaoka, Hasama, Yufu, Oita 879-5593, KEYWORDS
Japan.
Email: [email protected] Chikungunya virus, molecular diagnosis, RT-LAMP

Funding information

Lotte Foundation; GlaxoSmithKline
foundation; Suzuken Memorial Foundation;
Kurozumi Medical Foundation; Japan
Society for the Promotion of Science,
Grant/Award Number: 17H04649 ,
17K08889, 17K15680, 17K17104 and
18K16155

Communicated by: Tohru Ishitani

Parts of this study were presented at the 42nd annual Meeting of the Molecular Biology Society of Japan, December 3–6, Fukuoka, Japan.

© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd |wileyonlinelibrary.com/journal/gtc    1
Genes Cells. 2020;00:1–11. 

|2       Genes to Cells SAECHUE et al.

1  | INTRODUCTION a simple heat block, (b) it can skip both RNA extraction and
reverse transcription steps, and (c) the confirmation of ampli-
Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus fication of nucleic acids does not require electrophoresis and
belonging to the Togaviridae family (Schuffenecker gel imaging.
et  al.,  2006). CHIKV has a single-stranded positive RNA
genome, which encodes the capsid viral proteins, phos- Based on the LAMP method, a commercial RT-LAMP re-
pholipid envelopes and other components, and is approxi- agent kit, Loopamp® RNA amplification kit (Eiken Chemical
mately 11.8  kb in length (Khan, Morita, Mdel, & Hasebe, Co LTD), has been developed (Mori, Nagamine, Tomita, &
Mathenge, & Igarashi 2002). The virus is transmitted to hu- Notomi,  2001; Nagamine, Hase, & Notomi,  2002; Notomi
mans by CHIKV-infected Aedes aegypti and Aedes albopic- et al., 2000). This is a rapid and highly sensitive RT-LAMP
tus mosquitoes (Weaver, 2006). CHIKV was first described kit, but it is too expensive for testing a large number of sam-
in Tanzania in 1952 (Lumsden,  1955; Robinson,  1955; ples, particularly in low-income countries, and still relies on
Ross, 1956), followed by the first Asian outbreak reported in the use of a cold chain for enzyme storage. Although there are
1958 in Bangkok, Thailand. CHIKV is a re-emerging patho- some reports regarding RT-LAMP systems for CHIKV using
gen that can significantly impact public health in many trop- the Loopamp® kit, we aim to establish a CHIKV-specific RT-
ical African and Asian countries. CHIKV can equally affect LAMP system, which enables us to carry out on-site diagno-
both sexes at any age. The incubation period varies between 3 sis without a cold chain in a cost-effective manner.
and 7 days. In the acute phase of the infection, patients com-
monly present with high fever, severe arthralgia, skin rash 2  | RESULTS
and headache (Schwartz & Albert, 2010; Staples, Breiman,
& Powers, 2009). In particular, severe joint pain can impact 2.1  |  CHIKV RT-LAMP sensitivity
quality of life on a long-term basis.
To develop a new CHIKV-specific RT-LAMP system, we
Several diagnostic tests have been developed to detect designed seventeen different combinations of four or six
CHIKV infection in the acute or later stage of the disease. primers targeting the E1 or nonstructural protein 1 region of
Viral culture has traditionally been regarded as the gold stan- CHIKV using PrimerExplorer V5 software (Table  S1) and
dard for diagnosis of CHIKV, although the technique is cum- then compared the primer's sensitivity and specificity in the
bersome and requires a biosafety level 3 (BSL-3) laboratory. diagnosis of CHIKV. Although some primer sets showed
Serological tests are able to detect current and past infection low sensitivity and others resulted in nonspecific amplifica-
with CHIKV and estimate viral titers in the patient; how- tion (data not shown), we eventually selected a combination
ever, cross reaction with other alphaviruses is problematic of six primers, described in “Experimental procedures” and
(Blackburn, Besselaar, & Gibson,  1995; Karabatsos,  1975; evaluated the primer sets in further experiments.
Powers, Brault, Tesh, & Weaver,  2000). Nucleic acid am-
plification methods such as reverse transcriptase-poly- First, to evaluate the sensitivity of the RT-LAMP system
merase chain reaction (RT-PCR) and real-time PCR can be for CHIKV, the detection limit of the system was determined
reliable diagnostic methods with high sensitivity and speci- by serial dilution of Chikungunya viral culture supernatant.
ficity. However, those methods require high skills, appropri- The virus titer was determined in advance by plaque assay
ate facilities and expensive devices such as thermocyclers, in Vero cells (Figure S1). As shown in Figure  1a, the sen-
real-time PCR machines and gel documentation systems sitivity was monitored by visual inspection. The reaction
(Hasebe et  al.,  2002; Telles, Le Roux, Grivard, Vernet, & tubes containing 1,000, 200 and 40 PFU of CHIKV exhib-
Michault, 2009). ited a positive green-yellow fluorescence signal under a
blue-green (λ = 470 nm) LED light, but the tube containing
Recently, several isothermal amplification techniques 8 PFU of CHIKV failed to do so within a 20-min time frame
have been developed to overcome the limitations of con- (Figure 1a, left panel). The same trend was observed when a
ventional methods (Fire & Xu,  1995; Notomi et  al.,  2000; positive reaction was judged by a color change of HNB from
Piepenburg, Williams, Stemple, & Armes,  2006; Vincent, violet to sky blue (Figure 1a, right panel). The LAMP reac-
Xu, & Kong,  2004). Among them, reverse transcription tion was, however, positive for the 8 PFU of CHIKV sample
loop-mediated isothermal amplification (RT-LAMP) is a after 30 min of incubation. There were no false-positive re-
rapid, simple and highly sensitive isothermal gene amplifica- sults in the negative control even after an extended incubation
tion technique using a set of four specific primers to amplify period (up to 60 min).
the target RNA sequence, which can be enhanced by addi-
tional two loop primers (Nie et al., 2012; Nyan et al., 2014; Next, to quantify the fluorescent intensity in the RT-
Safavieh et  al.,  2016). RT-LAMP offers a remarkable ad- LAMP reaction, the GelGreen signals were measured by a
vantage over the conventional RT-PCR method because (a) real-time PCR machine. The gene amplification of CHIKV
the amplification is carried out at isothermal conditions on samples reached a plateau phase within 30 min, whereas the

SAECHUE et al. (a) CHIKV Genes to Cells |     3

F I G U R E 1   Sensitivity of the RT- PFU/tube NC CHIKV NC
LAMP monitored by (a) visual inspection 10 min
using GelGreen fluorescence dye (left) 20 min 12,00000 0 00 00 0 12,4000000 80 0 00 00
and HNB (right) and (b) a real-time 30 min 40 8
PCR machine, LightCycler™ 96; Roche 60 min 8
Diagnostics, using fluorescence signal of
GelGreen. The culture supernatant was (b) 18
directly applied by the RT-LAMP without
RNA extraction. (c) The RT-LAMP Fluorescence (10– 2) 12
products were separated by electrophoresis
using 1% agarose gel, stained with ethidium
bromide. (d) RT-PCR was performed by
using an extracted RNA from the same
CHIKV. The PCR products were observed
on a 1% agarose gel electrophoresis stained
with ethidium bromide. NC: negative
control (RNase free water), M: DNA marker

6

0 0 10 20 30 40 50 60 70 80 90
Time (min)

(c) CHIKV NC (d) CHIKV NC

PFU/tube 12,4000000 0 PFU/tube12,4000000 0 M
8 0 0

300bp

RT-LAMP reaction in the negative controls failed to reach the virus (RRV). The specific amplification of the RT-LAMP
log phase of amplification and remained in the background reactions was confirmed by direct visual inspection using
(Figure 1b). A positive reaction was confirmed by gel elec- the relevant indicators. The RT-LAMP system was able to
trophoresis which showed ladder-like patterns (Figure  1c). detect all CHIKV strains tested, and upon inspection at the
Moreover, we compared the sensitivity of the RT-LAMP 60-min mark, no cross reaction with other tested viruses was
system with that of RT-PCR targeting the E1 region using seen (Figure 2a). Moreover, we performed agarose gel elec-
purified RNA. Although intense bands were observed in the trophoresis to confirm DNA amplification (Figure  2b) and
lanes that contained 1,000 and 200 PFU of CHIKV, very faint the results indicated that our RT-LAMP system was able to
bands were detected in the 40 and 8 PFU of CHIKV lanes specifically amplify CHIKV strains. Finally, we determined
(Figure  1d). Taken together, the sensitivity of the CHIKV- the specificity of the RT-LAMP system using higher viral
specific RT-LAMP system is as great as that of conventional loads. The results showed that the system selectively detected
CHIKV-specific RT-PCR. CHIKV but not other viruses even in the highest viral load
dilutions at 10,000 PFU after 60 min incubation (Figure 2c).
2.2  |  CHIKV RT-LAMP specificity
2.3  |  Detection of CHIKV genome from
Next, we examined the specificity of the RT-LAMP system
by comparing against a panel of mosquito-borne viruses in- serum and blood sample
cluding 4 strains of CHIKV (SL11131, SL10571, BaH306,
and S27), Zika virus (ZIKV), dengue virus serotype 4 We evaluated the ability of the RT-LAMP system to detect
(DENV4), Japanese encephalitis virus (JEV), and Ross River CHIKV in biological samples. Both wild-type and type I
IFN receptor 1 (IFNAR1)-deficient mice were infected with

|4       Genes to Cells CHIKV strains SAECHUE et al.

(a) CHIKV strains F I G U R E 2   Specificity of the RT-
LAMP system was determined using 200
NCJSBSSZDRILa2LEERK171VHNV1V03V150437611 PFU of 4 strains of CHIKV (SL11131,
10 min JSSBSZDRIL2aLEERK171VHNV1V03V150437611 SL10571, BaH306 and S27), ZIKV, DENV
20 min NC serotype 4, JEV and RRV. The specificity
30 min CHIKV strains of the RT-LAMP system was monitored
60 min by (a) visual inspection using GelGreen
fluorescence dye (left) and HNB (right). (b)
(b) The RT-LAMP products were separated
by electrophoresis using 1% agarose
M NC SBSSaL2L171H10315037611 gel, stained with ethidium bromide. (c)
ZIKV Specificity of the RT-LAMP system was
JDEEVNV4 determined using 10,000, 5,000, 1,000, and
RRV 200 PFU of CHIKV (SL11131), ZIKV,
DENV serotype 4, JEV and RRV at 60 min
of incubation. NC: negative control (RNase
free water), M: DNA marker

(c) 1152N0000C00000000
NC
PFU/tube 1152N0000C00000000
NC
CHIKV
ZIKV

DENV4
JEV
RRV

104 PFU of CHIKV per mouse via subcutaneous injection. is able to correctly detect CHIKV in the mouse serum and
RT-PCR showed that CHIKV viremia in wild-type mice blood.
was very low and the levels of viremia were undetectable in
most of these mice (data not shown). However, the viremia 2.4  |  Dry RT-LAMP for CHIKV detection
in IFNAR1-deficient mice was high enough to cause death
in all mice within 6 days postinfection (Figure S2). Thus, we To make the RT-LAMP system less dependent on cold
decided to use the serum and blood collected from CHIKV- chain transportation, we dried up all reagents for the reac-
infected IFNAR1-deficient mice at 4 days postinfection. The tion. First, to determine the detection limit, serial dilution
CHIKV-infected serum samples were directly analyzed by of Chikungunya viral stock was measured by the dry RT-
the RT-LAMP system. All samples showed a positive reac- LAMP. The sensitivity of the system was monitored by vis-
tion at 30 min (Figure 3a), whereas three out of five samples ual inspection. The tube containing 1,000 PFU of CHIKV
were found positive by RT-PCR (Figure  3b). Furthermore, reacted after 60  min of incubation and the tube containing
we determined that the detection limit of the RT-LAMP 200 PFU of CHIKV showed completely positive after 80 min
system was 105 PFU per reaction for CHIKV-infected mice of incubation (Figure 4a). However, the system failed to de-
serum (Figure 3c). tect 40 and 8 PFU of CHIKV.

Next, the blood samples were added directly to the RT- Next, we quantified the intensity of fluorescence by a
LAMP system. At 20  min, two samples showed positive, real-time PCR machine. The gene amplification of CHIKV
and by 90 min, all samples were positive (Figure 3d). Taken samples containing 1,000 and 200 PFU reached a plateau
together, these results indicate that the RT-LAMP system

SAECHUE et al. Genes to Cells |     5

F I G U R E 3   RT-LAMP detection of (a) Uninfected CHIKV Uninfected CHIKV
CHIKV-infected IFNAR1-deficient mice
at 4 days postinfection. (a) Two microliters 1 2 3 4 5 6 7 8 9 10NCPC 1 2 3 4 5 6 7 8 9 10 NCPC
of serum from either uninfected mice or
CHIKV-infected mice was applied on the 10 min
RT-LAMP, incubated for the indicated
time and then visualized using GelGreen 20 min
fluorescence dye (left) and HNB (right).
(b) RNA extracted samples from the same 30 min
serum were analyzed by RT-PCR. (c) The
detection limit of the RT-LAMP system was 60 min
determined using a 10-fold serial dilution of
CHIKV with the serum viral titers indicated. (b) Uninfected CHIKV
Visual inspections using GelGreen
fluorescence dye (left) and HNB (right) are M 1 2 3 4 5 6 7 8 9 10 NC PC
shown. (d) Whole blood samples from either
uninfected mice or CHIKV-infected mice 300 bp
were mixed with the lysis buffer described
in Materials and Methods, and tested with (c) CHIKV CHIKV
the RT-LAMP system. Visual inspections PFU/tube 106 105104103102 101NS PC 106 105104103102 101NS PC
using GelGreen fluorescence dye (left) and
HNB (right) are shown. 1–5: uninfected 10 min
mice, 6–10: CHIKV-infected mice, NC:
negative control (RNase free water), PC: 20 min
positive control (1,000 PFU of CHIKV),
NS: negative serum control, M: DNA 30 min
marker
60 min

(d) Uninfected CHIKV Uninfected CHIKV

1 2 3 4 5 6 7 8 9 10 NCPC 1 2 3 4 5 6 7 8 9 10 NCPC

10 min

20 min

30 min

40 min

50 min

60 min

90 min

phase within 80 min, whereas the samples containing 40 and system was unable to detect CHIKV in the serum (data not
8 PFU of CHIKV did not enter the log phase of amplification shown), the liquid RT-LAMP system detected the viral ge-
(Figure 4b). nome in nine out of ten RNA samples (90%) within 20 min
of incubation. However, the system did not react with RNAs
Finally, the sensitivity of the dry RT-LAMP was verified prepared from healthy human serum (Figure 5a). In the case
using serum samples of CHIKV-infected mice. The results of the dry RT-LAMP system, six out of ten RNA samples
showed an initial positive reaction being observed at 30 min (60%) were detected in 20  min of incubation. Seven RNA
of incubation, whereas all others, with the exception of one samples (70%) showed a positive reaction at 30  min, and
sample, showed a positive reaction at 70 min of incubation by 60 min of incubation, nine RNA samples (90%) reacted
(Figure 4c). These results showed that the dry RT-LAMP sys- positively (Figure 5b). Nevertheless, both liquid and dry RT-
tem was able to correctly detect the CHIKV genome without LAMP systems have potential utility for on-site diagnosis of
a nonspecific reaction, although with less sensitivity than the CHIKV infection using purified RNA from human clinical
liquid RT-LAMP system. samples.

2.5  |  RT-LAMP with clinical samples 3  | DISCUSSION

To evaluate the RT-LAMP system as a screening kit, both CHIKV is a causative agent of Chikungunya fever, which
liquid and dry RT-LAMP systems were validated using is characterized by high fever and severe arthralgia.
CHIKV-infected patient samples. Although the RT-LAMP

|6       Genes to Cells SAECHUE et al.

(a) CHIKV NC CHIKV NC F I G U R E 4   The assessment of the dry
RT-LAMP system was performed based
PFU/tube Fluorescence (10–2) on (a) visual inspection and (b) real-time
50 min 12,4000000 monitoring as shown in Figure 1. (c) The
60 min 8 dry RT-LAMP system was assessed using
70 min 0 serum collected from uninfected mice or
80 min 0 CHIKV-infected IFNAR1-deficient mice
0 as shown in Figure 3. 1–5: uninfected mice
(b) 16 0 serum, 6–10: CHIKV-infected mice serum,
12,4000000 NC: negative control (RNase free water),
8 PC: positive control (1,000 PFU of CHIKV)
0
12 01,000 PFU/tube
0200 PFU/tube
040 PFU/tube
8 PFU/tube
8 0 PFU/tube
0 PFU/tube
0 PFU/tube NC

4 0 PFU/tube

0
0 10 20 30 40 50 60 70 80 90
Time (min)

(c) Uninfected CHIKV Uninfected CHIKV

1 2 3 4 5 6 7 8 9 10 NC PC 1 2 3 4 5 6 7 8 9 10 NC PC

10 min

30 min
40 min

60 min

70 min

Although a substantial number of infected patients remain previous reports regarding RT-LAMP methods for CHIKV
completely  asymptomatic, these patients can be a pub- detection (Lu et al., 2012; Parida et al., 2007), the reaction of
lic health concern as a potential source of transmission. RT-LAMP was monitored by turbidity using an expensive re-
Moreover, CHIKV and other arbovirus such as Zika virus, al-time turbidimeter. Goto et al. reported a colorimetric assay
dengue virus, and Ross River virus share the same vec- of the LAMP reaction using hydroxy naphthol blue (HNB),
tor species, geographical distribution, seasonal correlation which enables us to monitor gene amplification easily with
and clinical symptoms, which increases the difficulty of the naked eye. HNB is a metal ion indicator that can detect
species-specific diagnosis (Kraemer et  al.,  2015; Renault decreased Mg2+ concentration in accordance with the prog-
et  al.,  2007; Weaver & Reisen,  2010). Thus, it is neces- ress of the RT-LAMP reaction. For blood samples, however,
sary for public health laboratories to have access to an in- it is difficult to judge the positive or negative result of the
creased availability of proper diagnostic tests. Molecular LAMP reaction by the colorimetric assay because of the
PCR-based methods that detect pathogen-derived nucleic color of red blood cells (Figure 3d, right). The use of fluo-
acids are very sensitive and relatively rapid compared to rescent indicators such as SYBR Green and calcein has been
conventional culturing methods; however, PCR-based de- applied for the RT-LAMP reaction (Goto, Honda, Ogura,
tection of pathogens requires high skills, appropriate facili- Nomoto, & Hanaki, 2009; Parida et al., 2007). However, the
ties and expensive devices. assay using the intercalating dye such as SYBR Green is as-
sociated with an increased risk of contamination because the
The RT-LAMP system is a low-cost alternative diagnos- assay requires opening the tubes to add the dye to the reac-
tic tool, especially in resource-limited settings, because it tion after amplification. A metal ion indicator, calcein, can
does not require sophisticated equipment such as a thermo- be added to the prereaction solution of RT-LAMP; however,
cycler. It could be applied in the early diagnosis of CHIKV the brightness of calcein fluorescence is significantly weaker
infection because of its sensitivity, speed and simplicity. In

SAECHUE et al. Genes to Cells |     7

(a) Serum of CHIKV-infected patients GelGreen and HNB indicators instead of SYBR Green I.
1 2 3 4 5 6 7 8 9 10 NC PC Both their primer set and our primer set showed a highly sen-
sitive reaction with CHIKV in the Loopamp® kit; however,
10 min after an hour incubation, all negative controls began to show
false-positive reactions, probably due in part to a high con-
20 min centration of Mg2+ in the kit (data not shown). Accordingly,
the conditions of each reaction mixture must be customized
1 Serum of healthy donors 10 NC PC precisely for each pathogen.
60 min 2 3 45 67 8 9
More recently, related studies have been reported (Lopez-
(b) Serum of CHIKV-infected patients Jimena et al., 2018 and Hayashida et al., 2019). Thus, we
1 2 3 4 5 6 7 8 9 10 NC PC compared the sensitivity and specificity of our RT-LAMP
system to those of their systems. Hayashida et al. used the
10 min primer sequences that are identical to those used in the report
by Parida et al. Although their primer set seemed to show
20 min relatively higher sensitivity than our primer set in the LAMP
buffer we used, it showed the false positive at 60 min of incu-
30 min bation. However, our primer set did not show any false-pos-
itive reactions even after 120-min incubation. Lopez-Jimena
40 min et al. designed three distinct primer sets and all of them
were less sensitive than our primer set (data not shown).
60 min Accordingly, our primer set appears to be enough sensitive to
detect CHIKV without false-positive results.
F I G U R E 5   Extracted RNA from either CHIKV-infected patients
or healthy donor serum samples was evaluated by liquid RT-LAMP The development of the RT-LAMP system for on-site
system (a). The dry RT-LAMP system was assessed by using CHIKV- diagnosis requires a system effective enough to detect the
infected patients (b). Samples were incubated for the indicated time pathogen in clinical samples such as serum and blood. It is
and then visualized using GelGreen fluorescence dye. 1–10: extracted noteworthy that we evaluated our RT-LAMP system using
RNA, NC: negative control (RNase free water), PC: positive control blood samples from mice infected with CHIKV, in which the
(1,000 PFU of CHIKV) viral load was precisely controlled. When we used the serum
of CHIKV-infected IFNAR1-deficient mice, RT-LAMP was
than that of SYBR green fluorescence (Goto et  al.,  2009). able to detect the virus in the serum in just 30 min without
Furthermore, both fluorescent indicators are expensive for an additional RNA extraction step (Figure  3a). However, it
large-scale screening in CHIKV-endemic areas. To this end, took more time in the case of the blood samples (Figure 3d),
the development of a cost-effective diagnostic system based probably because of the blood samples being diluted 10
on RT-LAMP technology to detect CHIKV is desired. times with lysis buffer to inhibit coagulation and sup-
press biological inhibitors such as hemoglobin (Al-Soud &
In this study, we designed new LAMP primers targeting Radstrom,  2001). The sensitivity of the RT-LAMP system
the E1 region of CHIKV and used GelGreen and HNB as was 104 times lower in mice serum than in the viral culture.
fluorescent and colorimetric indicators, respectively. The Moreover, the RT-LAMP system failed to detect CHIKV in
RT-LAMP system clearly detected at least 8 PFU of CHIKV the serum from infected patients (data not shown) that re-
within 30  min by visual inspection, which was as great as quires an improvement in sensitivity of the system for biolog-
that of conventional CHIKV-specific RT-PCR. GelGreen is a ical samples. Hemoglobin and immunoglobulin G are known
cost-effective indicator, costing approximately one third the as PCR inhibitors in the blood; that is, hemoglobin directly
amount of SYBR Green or calcein, and can be added to the inhibits DNA polymerase activity and quenches the fluores-
prereaction solution of RT-LAMP like calcein. Moreover, cence of free dye molecules, and immunoglobulin G binds to
the fluorescence signal from GelGreen can be applied for single-strand genomic DNA leading to low amplification ef-
quantitative monitoring, if necessary, using a real-time PCR ficiency (Al-Soud & Radstrom, 2001; Sidstedt et al., 2018).
machine. A process for removing or suppressing these inhibitory fac-
tors might be necessary.
In a previous study, Parida et al. established the CHIKV
RT-LAMP method using a commercial based RT-LAMP, Another positive aspect of the development of the RT-
Loopamp® kit and SYBR Green I dye (Parida et al., 2007). LAMP system for on-site diagnosis is its portability in en-
To follow their study, we performed RT-LAMP under the demic areas. To make the RT-LAMP system less dependent
same conditions as Parida et al. used the Loopamp® kit with on a cold chain, we developed the dry RT-LAMP system
using trehalose as a potent protectant, which prevents the

|8       Genes to Cells SAECHUE et al.

enzyme from desiccation or freezing damage during the 4.3  |  Viral sample preparation
vitrification process of the reagents (Teramoto, Sachinvala,
& Shibata,  2008). Although the sensitivity of the dry RT- To determine the viral titer, plaque assays were performed as de-
LAMP system was lower than that of the liquid RT-LAMP scribed previously (Kamiyama et  al.,  2017). Briefly, confluent
system, the dry system was able to detect at least 200 PFU Vero cells in 12-well culture plates were infected with serial dilu-
of CHIKV in the culture supernatant as well as the virus in tions of each virus in 2% complete MEM. After 1-hr adsorption at
mouse serum at an 80% detection rate within 70 min, without 37°C, 2% methylcellulose (MP Biomedicals, Aurora, OH, USA)
a nonspecific reaction. Moreover, the system successfully de- was layered on the cells. Subsequently, cells were incubated for
tected viral RNA extracted from human clinical samples with 5 days at 37°C, 5% CO2 in a humidified environment, fixed in
a high detection rate (90%) (Figure 5). To improve the sensi- 10% formalin (FUJIFILM Wako Pure Chemical) and then stained
tivity of the dry system, a combination of different kinds of with 1.125% methylene blue (FUJIFILM Wako Pure Chemical)
saccharides for the vitrification process should be examined. to count the plaques and the plates were photographed.

We established a novel RT-LAMP system to detect the E1 IFNAR1-deficient mice were injected with 104 plaque
region of CHIKV, which accurately identifies the virus in a forming units (PFU) of CHIKV via subcutaneous injection.
cost-effective manner, without a cold chain. The system has The blood was collected from the mice at 4 days postinfec-
great potential to be applied for CHIKV diagnosis in endemic tion. 20 μl of whole blood samples were mixed with 180 μl
areas. of lysis buffer (0.1% Triton X-100 in RNase free water) for
the RT-LAMP assay. For the preparation of serum samples,
4  | EXPERIMENTAL whole blood was centrifuged at 13,000 g for 10 min and then
the supernatant was collected.
PROCEDURES
CHIKV-infected human samples and healthy human sam-
4.1  |  Virus and cells ples were collected at the Institute of Epidemiology, Disease
Control and Research (IEDCR), Dhaka, Bangladesh, and
Four strains of CHIKV (SL11131, SL10571, BaH306 Department of Gastroenterology, Faculty of Medicine, Oita
and S27), ZIKV strain PRVABC, DENV4, JEV and RRV University, Oita, Japan, respectively. CHIKV infection was
were kindly provided by Dr. Tomohiko Takasaki (National confirmed by conventional RT-PCR assay prior to RT-LAMP
Institute of Infectious Diseases). C6/36 cells (Aedes albop- assay. All experiments using human samples in this study
ictus) were maintained at 28°C in 5% CO2 in 10% com- were approved by the Ethics Committee of Oita University
plete MEM, which consisted of Eagle's MEM (Nissui, (approval no. 1691).
Tokyo, Japan) with 2  mM L-alanyl-glutamic acid (Gibco
GlutaMAX; Thermo Fisher Scientific, Waltham, MA, The genomic viral RNA was extracted from viral stock,
USA), 0.1  mM nonessential amino acids (Thermo Fisher blood and serum samples using a QuickGene RNA Tissue
Scientific), 0.15% sodium bicarbonate (Thermo Fisher Kit SⅡ (RT-S2; KURABO, Osaka, Japan) according to the
Scientific) and 10% FBS (Hyclone FBS; Thermo Fisher manufacturer's protocol. The RNA was eluted from the col-
Scientific). Vero cells (African Green Monkey Kidney umns at a final volume of 100 μl by elution buffer and was
Epithelial Cells) were maintained at 37°C in 5% CO2 in 10% stored at −80°C until being used for RT-PCR.
complete DMEM containing 55 µM 2-Mercaptoethanol
(Thermo Fisher Scientific), 100 U/mL Penicillin, 100 µg/ml 4.4  | RT-PCR
Streptomycin (Nacalai tesque, Kyoto, Japan) and 10% FBS.
CHIKV was subsequently propagated on either C6/36 cells RT-PCR was carried out using SuperScript III One-Step RT-
or Vero cells in 2% complete MEM. PCR System, Platinum Taq (Thermo Fisher Scientific) ac-
cording to the manufacturer's protocol. Reverse transcription
4.2  | Mice was performed for 30 min at 50°C and for 2 min at 94°C, fol-
lowed by PCR amplification with CHIKV E1-specific prim-
Male IFNAR1-deficient mice (Muller et  al.,  1994) aged ers (Lim et al., 2009) (Table 1). The amplification program
7–9 weeks were used throughout this study. Mice were kept was as follows: 35 cycles of 94°C for 30 s, 53°C for 30 s, and
in a biosafety level 3 (BSL-3) laboratory in the Division of 68°C for 1 min and a final extension step at 68°C for 2 min.
Laboratory Animal Science of Oita University (Oita, Japan).
All experiments using these mice were approved by and per- 4.5  | RT-LAMP
formed according to the guidelines of the Oita University
Animal Ethics Committee. The RT-LAMP primers were designed based on the se-
quences of the envelope protein 1 (E1) region of CHIKV

SAECHUE et al. Genes to Cells |     9

T A B L E 1   RT-LAMP and RT-PCR Primer Length Oligonucleotide sequence (5’ to 3’) Product
primers for Chikungunya virus (CHIKV) RT-LAMP (bp) size
CACACTGTGAGCGCGTAC
CHIKV-F3 18 GACGGTTTTGTATTCGCACG 300 bp
CHIKV-B3 20 AGCCCGGTCTGTTGACTAGAGTttttACACG
CHIKV-FIP 46 TAACAGTGATCCCGA
ACAGCCCCATGGTACTGGAGAttttTCAAGC
CHIKV-BIP 43 GATAGCGTTGGC
ACGGTACTCCCACCGTGT
CHIKV-FLP 18 GGAGCTACTGTCAGTCACTTTG
CHIKV-BLP 22
RT-PCR ACGCAATTGAGCGAAGCACAT
10294f 21 AAATTGTCCTGGTCTTCCTG
10573r 20

strain S27-African prototype (GenBank accession number For the primer dry-up step, 100 μM stocks of 6 primers
AF369024). A set of six RT-LAMP primers including two were prepared in RNase free water. Primer mixtures con-
inner primers (FIP and BIP), two outer primers (F3 and B3) sisting of 0.4  μl each of FIP and BIP, 0.05  μl each of F3
and two loop primers (FLP and BLP) were designed with and B3, and 0.2 μl each of FLP and BLP, 0.9 μl of 2 M tre-
PrimerExplorer V5 software (http://primer​explo​rer.jp/e/) halose, 0.14 μl of 50% glycerol (Nacalai tesque) and 0.5 μl
(Table 1). TTTT spacers were added to FIP and BIP. of indicator were mixed and placed into the bottom of the
microtube. The final primer concentrations of each primer
The RT-LAMP reaction was carried out in a total of 25 μl in the reaction were 1.6 μM (FIP and BIP), 0.2 μM (F3 and
reaction volume containing 2  μl of viral culture superna- B3) and 0.4 μM (FLP and BLP).
tant, 1 μl of 25X LAMP buffer (500 mM Tris-HCl [pH 8.8],
250 mM KCl and 25 mM MgSO4), 6 mM MgSO4 (final con- For the final dry-up step, the mixture was dried for 6 hr
centration 7 mM), 1.4 mM dNTPs (25 mM each; NIPPON under a flow of clean air. The tubes were further dried in
GENE CO., LTD.), eight units Bst DNA Polymerase (8  U/ a desiccator with phosphorus oxide and silica gel overnight
μl; NIPPON GENE CO., LTD.), four units RNase inhibitor under a vacuum until completely dried. After this step, the
(40 U/μl; Nacalai tesque), 0.6 units AMV reverse transcrip- tubes were placed in an aluminum bag with zeolite molecular
tase (20 U/μl; NIPPON GENE CO., LTD.), 0.2 M trehalose sieves, tightly sealed, and stored until use. The reaction tubes
(FUJIFILM Wako Pure Chemical), 1.6 μM each of FIP and were reconstituted with 25  μl of LAMP buffer containing
BIP, 0.2  μM each of F3 and B3, 0.4  μM each of FLP and viral template, 6  mM MgSO4 and 0.1% Triton X-100. The
BLP, 1 μl of indicators (3 mM hydroxynaphthol blue [HNB; reaction tubes were inverted several times under vigorous ag-
MP Biomedicals], 0.35% v/v GelGreen [10,000X in DMSO; itation until the dried reagents were completely reconstituted.
Biotium, Inc.]) and 0.1% Triton X-100 in DDW were added
to make a 25-μl reaction mix. The RT-LAMP reaction mix- 4.7  |  Monitoring of amplification by the RT-
ture was incubated at 59°C for 60 min.
LAMP
4.6  |  Drying RT-LAMP reagents procedure
For visual inspection, the amplified DNA was stained with
The procedure for drying RT-LAMP was described previ- GelGreen fluorescent dye and observed under a blue-green
ously (Hayashida, Kajino, Hachaambwa, Namangala, & (λ = 470 nm) LED light. The color of the CHIKV-positive
Sugimoto,  2015). Briefly, for the enzyme and dNTP dry- samples exhibited green-yellow fluorescence in accordance
up step, 1.6 μl of 2 M trehalose, 1.4 μl of dNTPs (25 mM with an RT-LAMP reaction. In addition, the color of the posi-
each), 0.05  μl of Bst DNA Polymerase (120  U/μl; New tive samples changed from violet to sky blue by HNB under
England Biolabs), 0.25  μl of Bst DNA Polymerase (8  U/ ambient light conditions.
μl), 0.1 μl of RNase inhibitor (40 U/μl), 0.04 μl of AMV
reverse transcriptase (20 U/μl) and 0.5 μl of indicator were The amplification in RT-LAMP reactions was monitored
mixed and placed in the center of a microtube cap (Watson at 59°C using a real-time PCR machine (LightCycler™96;
Bio Lab). Roche Diagnostics, Mannheim, Germany). Fluorescence sig-
nal of GelGreen was automatically recorded every 1 min for
90 min. After amplification by RT-LAMP, the final products

|10       Genes to Cells SAECHUE et al.

were analyzed by agarose gel electrophoresis on a 1% gel in Kamiyama, N., Soma, R., Hidano, S., Watanabe, K., Umekita, H.,
1X Tris acetate-EDTA buffer (0.04  M Tris acetate, 1  mM Fukuda, C., … Kobayashi, T. (2017). Ribavirin inhibits Zika virus
EDTA), stained with ethidium bromide and visualized on a (ZIKV) replication in vitro and suppresses viremia in ZIKV-infected
UV transilluminator at 302 nm. STAT1-deficient mice. Antiviral Research, 146, 1–11. https://doi.
org/10.1016/j.antiv​iral.2017.08.007
ACKNOWLEDGMENTS
Karabatsos, N. (1975). Antigenic relationships of group A arboviruses
We thank Ms. Chiharu Aoki and Ms. Yoko Kudo for their by plaque reduction neutralization testing. The American Journal
excellent secretarial assistance and the members of Dr. of Tropical Medicine and Hygiene, 24(3), 527–532. https://doi.
Kobayashi's laboratory for valuable discussions. This work org/10.4269/ajtmh.1975.24.527
was supported by Grants-in-Aid from the Japan Society
for the Promotion of Science (Grant numbers 17K08889, Khan, A. H., Morita, K., Parquet Md Mdel, C., Hasebe, F., Mathenge,
17H04649 [TK], 18K16155 [TO], 17K17104 [NK] and E. G., & Igarashi, A. (2002). Complete nucleotide sequence of chi-
17K15680 [SH]), Suzuken Memorial Foundation, Kurozumi kungunya virus and evidence for an internal polyadenylation site.
Medical Foundation and GSK Japan Research Grant 2016. Journal of General Virology, 83(Pt 12), 3075–3084. https://doi.
Lotte Foundation. org/10.1099/0022-1317-83-12-3075

CONFLICT OF INTEREST Kraemer, M. U. G., Sinka, M. E., Duda, K. A., Mylne, A. Q. N., Shearer,
The authors have no competing financial interests to declare. F. M., Barker, C. M., … Hay, S. I. (2015). The global distribution
of the arbovirus vectors Aedes aegypti and Ae. albopictus. Elife, 4,
ORCID e08347. https://doi.org/10.7554/eLife.08347

Takashi Kobayashi  https://orcid. Lim, C.-K., Kotaki, A., Nishibori, T., Ito, M., Watanabe, K., Tanaka,
org/0000-0002-9069-5736 K., … Takasaki, T. (2009). Chikungunya virus isolated from a
returnee to Japan from Sri Lanka: Isolation of two sub-strains
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L., … Pierre, V. (2007). A major epidemic of chikungunya virus
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pathogenic properties and relationship to the epidemic. The Journal Additional supporting information may be found online in
of Hygiene, 54(2), 177–191. the Supporting Information section.

Safavieh, M., Kanakasabapathy, M. K., Tarlan, F., Ahmed, M. U., Zourob, How to cite this article: Saechue B, Kamiyama N,
M., Asghar, W., & Shafiee, H. (2016). Emerging Loop-Mediated Wang Y, et al. Development of a portable reverse
Isothermal Amplification-Based Microchip and Microdevice transcription loop-mediated isothermal amplification
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& Engineering, 2(3), 278–294. https://doi.org/10.1021/acsbio​ mate​ in a cost-effective manner. Genes Cells.
rials.5b00449 2020;00:1–11. https://doi.org/10.1111/gtc.12797

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| |Received: 27 December 2019    Revised: 1 June 2020    Accepted: 10 June 2020 Genes to Cells

DOI: 10.1111/gtc.12797

ORIGINAL ARTICLE

Development of a portable reverse transcription loop-mediated
isothermal amplification system to detect the E1 region of
Chikungunya virus in a cost-effective manner

Benjawan Saechue1  | Naganori Kamiyama1  | Yinan Wang1  | Chiaki Fukuda1  |
Kei Watanabe1  | Yasuhiro Soga1  | Mizuki Goto1  | Astri Dewayani1  | Shimpei Ariki1  |
Haruna Hirose1  | Sotaro Ozaka1  | Nozomi Sachi1  | Shinya Hidano1  | Khaledul Faisal2  |
Rajashree Chowdhury2  | Md. Anik Ashfaq Khan2  | Faria Hossain2  | Prakash Ghosh2  |
Tahmina Shirin3  | Dinesh Mondal2  | Kazunari Murakami4  | Takashi Kobayashi1

1Department of Infectious Disease Control, Abstract
Faculty of Medicine, Oita University, Oita,
Japan Chikungunya fever is a mosquito-borne disease cause of persistent arthralgia. The
2Nutrition and Clinical Services Division, current diagnosis of Chikungunya virus (CHIKV) relies on a conventional reverse
International Centre for Diarrhoeal Disease transcription polymerase chain reaction assay. Reverse transcription loop-mediated
Research, Bangladesh (icddr, b), Dhaka, isothermal amplification (RT-LAMP) is a rapid and simple tool used for DNA-based
Bangladesh diagnosis of a variety of infectious diseases. In this study, we established an RT-
3Department of Virology, Institute of LAMP system to recognize CHIKV by targeting the envelope protein 1 (E1) gene
Epidemiology Disease Control and that could also detect CHIKV at a concentration of 8 PFU without incorrectly detect-
Research (IEDCR), Dhaka, Bangladesh ing other mosquito-borne viruses. The system also amplified the E1 genome in the
4Department of Gastroenterology, Faculty serum of CHIKV-infected mice with high sensitivity and specificity. Moreover, we
of Medicine, Oita University, Oita, Japan established a dry RT-LAMP system that can be transported without a cold chain,
which detected the virus genome in CHIKV-infected patient samples with high ac-
Correspondence curacy. Thus, the dry RT-LAMP system has great potential to be applied as a novel
CHIKV screening kit in endemic areas.
Takashi Kobayashi, Oita University, 1-1
Idaigaoka, Hasama, Yufu, Oita 879-5593, KEYWORDS
Japan.
Email: [email protected] Chikungunya virus, molecular diagnosis, RT-LAMP

Funding information

Lotte Foundation; GlaxoSmithKline
foundation; Suzuken Memorial Foundation;
Kurozumi Medical Foundation; Japan
Society for the Promotion of Science,
Grant/Award Number: 17H04649 ,
17K08889, 17K15680, 17K17104 and
18K16155

Communicated by: Tohru Ishitani

Parts of this study were presented at the 42nd annual Meeting of the Molecular Biology Society of Japan, December 3–6, Fukuoka, Japan.

© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd |wileyonlinelibrary.com/journal/gtc    1
Genes Cells. 2020;00:1–11. 

|2       Genes to Cells SAECHUE et al.

1  | INTRODUCTION a simple heat block, (b) it can skip both RNA extraction and
reverse transcription steps, and (c) the confirmation of ampli-
Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus fication of nucleic acids does not require electrophoresis and
belonging to the Togaviridae family (Schuffenecker gel imaging.
et  al.,  2006). CHIKV has a single-stranded positive RNA
genome, which encodes the capsid viral proteins, phos- Based on the LAMP method, a commercial RT-LAMP re-
pholipid envelopes and other components, and is approxi- agent kit, Loopamp® RNA amplification kit (Eiken Chemical
mately 11.8  kb in length (Khan, Morita, Mdel, & Hasebe, Co LTD), has been developed (Mori, Nagamine, Tomita, &
Mathenge, & Igarashi 2002). The virus is transmitted to hu- Notomi,  2001; Nagamine, Hase, & Notomi,  2002; Notomi
mans by CHIKV-infected Aedes aegypti and Aedes albopic- et al., 2000). This is a rapid and highly sensitive RT-LAMP
tus mosquitoes (Weaver, 2006). CHIKV was first described kit, but it is too expensive for testing a large number of sam-
in Tanzania in 1952 (Lumsden,  1955; Robinson,  1955; ples, particularly in low-income countries, and still relies on
Ross, 1956), followed by the first Asian outbreak reported in the use of a cold chain for enzyme storage. Although there are
1958 in Bangkok, Thailand. CHIKV is a re-emerging patho- some reports regarding RT-LAMP systems for CHIKV using
gen that can significantly impact public health in many trop- the Loopamp® kit, we aim to establish a CHIKV-specific RT-
ical African and Asian countries. CHIKV can equally affect LAMP system, which enables us to carry out on-site diagno-
both sexes at any age. The incubation period varies between 3 sis without a cold chain in a cost-effective manner.
and 7 days. In the acute phase of the infection, patients com-
monly present with high fever, severe arthralgia, skin rash 2  | RESULTS
and headache (Schwartz & Albert, 2010; Staples, Breiman,
& Powers, 2009). In particular, severe joint pain can impact 2.1  |  CHIKV RT-LAMP sensitivity
quality of life on a long-term basis.
To develop a new CHIKV-specific RT-LAMP system, we
Several diagnostic tests have been developed to detect designed seventeen different combinations of four or six
CHIKV infection in the acute or later stage of the disease. primers targeting the E1 or nonstructural protein 1 region of
Viral culture has traditionally been regarded as the gold stan- CHIKV using PrimerExplorer V5 software (Table  S1) and
dard for diagnosis of CHIKV, although the technique is cum- then compared the primer's sensitivity and specificity in the
bersome and requires a biosafety level 3 (BSL-3) laboratory. diagnosis of CHIKV. Although some primer sets showed
Serological tests are able to detect current and past infection low sensitivity and others resulted in nonspecific amplifica-
with CHIKV and estimate viral titers in the patient; how- tion (data not shown), we eventually selected a combination
ever, cross reaction with other alphaviruses is problematic of six primers, described in “Experimental procedures” and
(Blackburn, Besselaar, & Gibson,  1995; Karabatsos,  1975; evaluated the primer sets in further experiments.
Powers, Brault, Tesh, & Weaver,  2000). Nucleic acid am-
plification methods such as reverse transcriptase-poly- First, to evaluate the sensitivity of the RT-LAMP system
merase chain reaction (RT-PCR) and real-time PCR can be for CHIKV, the detection limit of the system was determined
reliable diagnostic methods with high sensitivity and speci- by serial dilution of Chikungunya viral culture supernatant.
ficity. However, those methods require high skills, appropri- The virus titer was determined in advance by plaque assay
ate facilities and expensive devices such as thermocyclers, in Vero cells (Figure S1). As shown in Figure  1a, the sen-
real-time PCR machines and gel documentation systems sitivity was monitored by visual inspection. The reaction
(Hasebe et  al.,  2002; Telles, Le Roux, Grivard, Vernet, & tubes containing 1,000, 200 and 40 PFU of CHIKV exhib-
Michault, 2009). ited a positive green-yellow fluorescence signal under a
blue-green (λ = 470 nm) LED light, but the tube containing
Recently, several isothermal amplification techniques 8 PFU of CHIKV failed to do so within a 20-min time frame
have been developed to overcome the limitations of con- (Figure 1a, left panel). The same trend was observed when a
ventional methods (Fire & Xu,  1995; Notomi et  al.,  2000; positive reaction was judged by a color change of HNB from
Piepenburg, Williams, Stemple, & Armes,  2006; Vincent, violet to sky blue (Figure 1a, right panel). The LAMP reac-
Xu, & Kong,  2004). Among them, reverse transcription tion was, however, positive for the 8 PFU of CHIKV sample
loop-mediated isothermal amplification (RT-LAMP) is a after 30 min of incubation. There were no false-positive re-
rapid, simple and highly sensitive isothermal gene amplifica- sults in the negative control even after an extended incubation
tion technique using a set of four specific primers to amplify period (up to 60 min).
the target RNA sequence, which can be enhanced by addi-
tional two loop primers (Nie et al., 2012; Nyan et al., 2014; Next, to quantify the fluorescent intensity in the RT-
Safavieh et  al.,  2016). RT-LAMP offers a remarkable ad- LAMP reaction, the GelGreen signals were measured by a
vantage over the conventional RT-PCR method because (a) real-time PCR machine. The gene amplification of CHIKV
the amplification is carried out at isothermal conditions on samples reached a plateau phase within 30 min, whereas the

SAECHUE et al. (a) CHIKV Genes to Cells |     3

F I G U R E 1   Sensitivity of the RT- PFU/tube NC CHIKV NC
LAMP monitored by (a) visual inspection 10 min
using GelGreen fluorescence dye (left) 20 min 12,00000 0 00 00 0 12,4000000 80 0 00 00
and HNB (right) and (b) a real-time 30 min 40 8
PCR machine, LightCycler™ 96; Roche 60 min 8
Diagnostics, using fluorescence signal of
GelGreen. The culture supernatant was (b) 18
directly applied by the RT-LAMP without
RNA extraction. (c) The RT-LAMP Fluorescence (10– 2) 12
products were separated by electrophoresis
using 1% agarose gel, stained with ethidium
bromide. (d) RT-PCR was performed by
using an extracted RNA from the same
CHIKV. The PCR products were observed
on a 1% agarose gel electrophoresis stained
with ethidium bromide. NC: negative
control (RNase free water), M: DNA marker

6

0 0 10 20 30 40 50 60 70 80 90
Time (min)

(c) CHIKV NC (d) CHIKV NC

PFU/tube 12,4000000 0 PFU/tube12,4000000 0 M
8 0 0

300bp

RT-LAMP reaction in the negative controls failed to reach the virus (RRV). The specific amplification of the RT-LAMP
log phase of amplification and remained in the background reactions was confirmed by direct visual inspection using
(Figure 1b). A positive reaction was confirmed by gel elec- the relevant indicators. The RT-LAMP system was able to
trophoresis which showed ladder-like patterns (Figure  1c). detect all CHIKV strains tested, and upon inspection at the
Moreover, we compared the sensitivity of the RT-LAMP 60-min mark, no cross reaction with other tested viruses was
system with that of RT-PCR targeting the E1 region using seen (Figure 2a). Moreover, we performed agarose gel elec-
purified RNA. Although intense bands were observed in the trophoresis to confirm DNA amplification (Figure  2b) and
lanes that contained 1,000 and 200 PFU of CHIKV, very faint the results indicated that our RT-LAMP system was able to
bands were detected in the 40 and 8 PFU of CHIKV lanes specifically amplify CHIKV strains. Finally, we determined
(Figure  1d). Taken together, the sensitivity of the CHIKV- the specificity of the RT-LAMP system using higher viral
specific RT-LAMP system is as great as that of conventional loads. The results showed that the system selectively detected
CHIKV-specific RT-PCR. CHIKV but not other viruses even in the highest viral load
dilutions at 10,000 PFU after 60 min incubation (Figure 2c).
2.2  |  CHIKV RT-LAMP specificity
2.3  |  Detection of CHIKV genome from
Next, we examined the specificity of the RT-LAMP system
by comparing against a panel of mosquito-borne viruses in- serum and blood sample
cluding 4 strains of CHIKV (SL11131, SL10571, BaH306,
and S27), Zika virus (ZIKV), dengue virus serotype 4 We evaluated the ability of the RT-LAMP system to detect
(DENV4), Japanese encephalitis virus (JEV), and Ross River CHIKV in biological samples. Both wild-type and type I
IFN receptor 1 (IFNAR1)-deficient mice were infected with

|4       Genes to Cells CHIKV strains SAECHUE et al.

(a) CHIKV strains F I G U R E 2   Specificity of the RT-
LAMP system was determined using 200
NCJSBSSZDRILa2LEERK171VHNV1V03V150437611 PFU of 4 strains of CHIKV (SL11131,
10 min JSSBSZDRIL2aLEERK171VHNV1V03V150437611 SL10571, BaH306 and S27), ZIKV, DENV
20 min NC serotype 4, JEV and RRV. The specificity
30 min CHIKV strains of the RT-LAMP system was monitored
60 min by (a) visual inspection using GelGreen
fluorescence dye (left) and HNB (right). (b)
(b) The RT-LAMP products were separated
by electrophoresis using 1% agarose
M NC SBSSaL2L171H10315037611 gel, stained with ethidium bromide. (c)
ZIKV Specificity of the RT-LAMP system was
JDEEVNV4 determined using 10,000, 5,000, 1,000, and
RRV 200 PFU of CHIKV (SL11131), ZIKV,
DENV serotype 4, JEV and RRV at 60 min
of incubation. NC: negative control (RNase
free water), M: DNA marker

(c) 1152N0000C00000000
NC
PFU/tube 1152N0000C00000000
NC
CHIKV
ZIKV

DENV4
JEV
RRV

104 PFU of CHIKV per mouse via subcutaneous injection. is able to correctly detect CHIKV in the mouse serum and
RT-PCR showed that CHIKV viremia in wild-type mice blood.
was very low and the levels of viremia were undetectable in
most of these mice (data not shown). However, the viremia 2.4  |  Dry RT-LAMP for CHIKV detection
in IFNAR1-deficient mice was high enough to cause death
in all mice within 6 days postinfection (Figure S2). Thus, we To make the RT-LAMP system less dependent on cold
decided to use the serum and blood collected from CHIKV- chain transportation, we dried up all reagents for the reac-
infected IFNAR1-deficient mice at 4 days postinfection. The tion. First, to determine the detection limit, serial dilution
CHIKV-infected serum samples were directly analyzed by of Chikungunya viral stock was measured by the dry RT-
the RT-LAMP system. All samples showed a positive reac- LAMP. The sensitivity of the system was monitored by vis-
tion at 30 min (Figure 3a), whereas three out of five samples ual inspection. The tube containing 1,000 PFU of CHIKV
were found positive by RT-PCR (Figure  3b). Furthermore, reacted after 60  min of incubation and the tube containing
we determined that the detection limit of the RT-LAMP 200 PFU of CHIKV showed completely positive after 80 min
system was 105 PFU per reaction for CHIKV-infected mice of incubation (Figure 4a). However, the system failed to de-
serum (Figure 3c). tect 40 and 8 PFU of CHIKV.

Next, the blood samples were added directly to the RT- Next, we quantified the intensity of fluorescence by a
LAMP system. At 20  min, two samples showed positive, real-time PCR machine. The gene amplification of CHIKV
and by 90 min, all samples were positive (Figure 3d). Taken samples containing 1,000 and 200 PFU reached a plateau
together, these results indicate that the RT-LAMP system