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
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.
Result: 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.