Establishment Of Nested RT-PCR And Real-Time Fluorescence RT-PCR Methods To Detect West Nile Virus

West Nile Virus (WNV) is an arthropod-borne virus that belongs to the genus Flavivirus of the family Flaviviridae. WNV induces potentially lethal infections in humans and horses and is maintained in nature through a transmission cycle involving primarily Culex species mosquitoes and birds. Until 1999, the geographical distribution of WNV was restricted to Africa, the Middle East, western and central Asia, India, and Europe. Since the outbreak of WN encephalitis in humans and horses in New York City in late August 1999, WNV has spread throughout the United States. This outbreak was the first occurrence of WNV in the Western hemisphere and it has the trend to spread out. Currently, there are no effective therapies against WNV infection for use in humans. Therefore, it is important to establish detection methods to prevention of WNV invasion.1. Nested RT-PCR to detect WNVTwo pairs of nested RT-PCR primers were designed and synthesized based on the nucleotide sequences of WNV in GenBank. After optimization of the detection methods, they were used to amplify WNV inactivated vaccine. The results showed that all primers could get the specific bands. The high specificity of the methods was demonstrated by detecting 11 viruses, such as Japanese encephalitis virus, Yellow Fever virus, and so on. The maximal detection limits by two pairs of nested RT-PCR primers were approximately 85 copies and 62 copies of double-stranded DNAs, respectively. Therefore, two nested RT-PCR methods developed here for WNV detection had the characteristic of high efficiency, speediness, specificity and sensitivity. They can be used to entry-exit inspection.2. Real-time Fluorescence RT-PCR to detect WNVThree pairs of PCR primers and probes were designed and synthesized by DNAStar and Primer Expression 3.0 based on the nucleotide sequences of WNV in GenBank. After optimization of the detection methods, WNYA,WNYB and WNYC real-time fluorescence RT-PCR techniques were developed to amplify WNV. The high specificity of the methods was demonstrated by detecting 11 viruses, such as Japanese encephalitis virus, Yellow Fever virus, and so on. The maximal detection limits of WNYA and WNYC real-time fluorescence RT-PCR were approximately 30 copies and 65 copies of double-stranded DNAs, 420 copies and 460 copies of plasmid DNA, respectively, which were more sensitive than WNYB real-time fluorescence RT-PCR to cDNA. Therefore, WNYA and WNYC real-time fluorescence RT-PCR on the basis of the TaqMan technique developed here for WNV detection had the characteristic of high efficiency, speediness, specificity and sensitivity that could be used to detect WNV.3. Cloning the partly gene sequences of WNVThe study was used standard RT-PCR to amplify the intent product, which contained the genomic sequence of two pairs of nested RT-PCR external primers and two pairs of real-time fluorescence RT-PCR primers. In this research, pMD18-T cloning vector was used and the four intend product successfully obtained, which were named pMD18-T-A, pMD18-T-B, pMD18-T-YA and pMD18-T-YC. The accurate nucleotide sequence of the target genes were determined by sequencing and the pMD18-T-A, pMD18-T-B, pMD18-T-YA and pMD18-T-YC plasmids could be used as positive control for the methods established by us.4. Making up the detection kits of WNVTwo detection kits of WNV were made up by using established nested RT-PCR and real-time fluorescence RT-PCR methods and the result showed that the kits could be used to detect the WNV quickly after checking the effect of the kits.