Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Artificial synthesis of conserved segment s gene fragment of rift valley fever virus and preliminary study of its reverse transcription loop-mediated isothermal amplification detection method

Zexiao Yang¹ , Guili Li¹, Yihong Hou², Xueping Yao¹, Ranyang Ren¹, Houxun Ya¹, Shanzhen Peng¹, Xingyu Lin¹, Yin Wang¹

¹College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014; ²Changde Entry-Exit Inspection and Quarantine Bureau, Changde 415100, China.

For correspondence:- Zexiao Yang Email: yzxyang2003@126.com Tel:+86083502885077

Received: 21 June 2015 Accepted: 26 October 2015 Published: 27 December 2015

Citation: Yang Z, Li G, Hou Y, Yao X, Ren R, Ya H, et al. Artificial synthesis of conserved segment s gene fragment of rift valley fever virus and preliminary study of its reverse transcription loop-mediated isothermal amplification detection method. Trop J Pharm Res 2015; 14(12):2193-2200 doi: 10.4314/tjpr.v14i12.6

© 2015 The authors.
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited..

Abstract

Purpose: To develop a rapid detection method for Rift Valley fever virus (RVFV) diagnosis.

Methods: According to the reference sequences of RVFV published in GenBank, nine overlapping polymerase chain reaction (PCR) primers and four specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) primers were designed using DNAStar and LAMP primer design software, respectively. Based on the synthesis of a conserved part of the RVFV S segment gene sequence using overlapping PCR, RT-LAMP assay was first established and evaluated after a series of tests, including, optimization of reaction conditions, and sensitivity and specificity tests.

Results: A target RVFV S segment gene fragment of 288 bp was synthesised. The optimal reaction conditions for RT-LAMP assay were 63 °C for 45 min: the assay has a specific ladder-like pattern of amplification bands from about 120 bp. The lowest target gene copy number of RT-LAMP for RVFV detection was 70 copies. The assay showed good specificity as only the synthesised target RVFV gene was amplified with no amplification for the detection of Peste des petits ruminants virus, Epidemic encephalitis B virus, E. coli, Pasteurella multocida, or Salmonella.

Conclusion: This study provides a rapid, sensitive, specific RT-LAMP method for RVFV detection.

Keywords: Rift valley fever virus, Overlapping polymerase chain reaction, Reverse transcription loop-mediated isothermal amplification, Rapid diagnosis test