西尼罗病毒荧光RT-PCR检测方法的建立

目的用TaqMan技术建立荧光RT-PCR方法,用于检测西尼罗病毒(WNV)。方法从GenBank上调取WNV的基因序列,设计WNYA、WNYB、WNYC三套荧光RT-PCR,经优化后用于WNV灭活苗、乙型脑炎病毒(JEV)、黄热病毒(YFV)等11种病毒核酸的检测,同时用10倍倍比稀释的双链DNA、质粒DNA、cDNA、RNA进行扩增,以检测其灵敏度。结果建立的WNYA、WNYC荧光RT-PCR检出WNV灭活苗阳性,而JEV等10种非WNV病毒均为阴性,能检出30个拷贝和65个拷贝的双链DNA、420个拷贝和460个拷贝的质粒DNA;WNYB的反转录效率明显低于WNYA和WNYC。结论建立的WNYA、WNYC荧光RT-PCR可作为检测WNV的技术储备。

Detection of West Nile virus by real-time RT-PCR

Three pairs of primers used in the real-time PCR(WNYA,WNYB and WNYC) were designed and synthesized based on the nucleotide sequences of West Nile virus(WNV)in GenBank by TaqMan technique.After optimization,these primers were applied to detect the nucleic acids of 11 viruses,including WNV inactivated vaccine strain,Japanese B encephalitis virus(JEV),yellow fever virus(YFV),etc.Meanwhile,their sensitivity was determined by the amplifications of 10 times diluted double strand-DNA,plasmid DNA,cDNA and RNA.It was found that the maximal detection limits of WNYA and WNYC real-time PCR were approximately 30 copies and 65 copies for double strand-DNA;while those were 420 and 460 copies for plasmid DNA,respectively.which appeared to be more sensitive than that of WNYB real time PCR to cDNA.It is evident that the WNYA and WNYC real-time PCR on the basis of TaqMan technique developed for the detection of WNV shows high degree of specificity and sensitivity and can be used for WNV inspection and investigation.