Establishment Of Rapid Diagnostic Methods For Detection Of West Nile Virus By Nest RT-PCR And TaqMan Real-time RT-PCR

West Nile virus is taxonomically placed within the family Flaviviridae, genus Flavivirus. Within the genus Flavivirus, WNV has been serologically classified within the JEV antigenic complex, which includes the human pathogens JE, Murray Valley encephalitis, SLE and Kunjin viruses. It is a mosquito-borne virus, Culex species mosquitoes are main vectors. WNV can cause febrile illness, encephalitis and fatal meningoencephalitis in humans, horses and birds. Since its first isolating in 1937 in Uganda, it has become one of the most widespread f laviviruses worldwide and developed into a nationwide health and veterinary problem. West Nile fever is one of the list significant diseases of OIE. Although the virus has not been found in our country, it is great important to establish exact and rapid diagnostic methods for preventing the virus' s invading.In this article, RT-nested PCR, TaqMan RT-PCR assay methods were reported to be established for detection of WNV.In this study, the RT-nested PCR method to detect WNV nucleic acid was developed using a 1999 North American isolate, basing on the sequence of WNV E gene, RT-nPCR oligonucleotide primers were designed and their specificity were identified by Blast and PCR. RNA template was observed by transcription in vitro, after the first stage RT-PCR and the second stage nested PCR,WN E protein gene regions of 445bp and 248bp were amplified respectively. This method had excellent specificity and sensibility, 6X10° RNA copy per reactioncould be detected.TaqMan assay poses less risk of laboratory cross-contamination than RT-nested PCR. In this study, a TaqMan RT-PCR assay had a sensibility of 3X101 RNA copy per reaction was developed. Here we designed oligonucleotide primers and FAM-and-TAMRA-labeled WN virus specific probe by using the nucleotide sequence of the New York 1999 WN virus isolate too. RNA template was also observed by transcription in vitro. The TaqMan assay was compared to a traditional RT-PCR assay and proved to be specificity for WNV and demonstrated a greater sensitivity than the traditional RT-PCR method.