实时RT-PCR检测西尼罗病毒

目的建立西尼罗病毒快速、敏感和特异的实时RT-PCR法,用于西尼罗病毒疾病的早期诊断。方法对Vero细胞增殖的西尼罗病毒分别进行常规RT-PCR和实时RT-PCR法扩增,以PFU／ml为参考标准,比较二者的敏感性和特异性。并用实时RT-PCR法检测西尼罗病毒感染的小鼠组织标本。结果采用所建立的实时RT-PCR法和常规RT-PCR法可分别检测到0．02PFU／ml和2PFU／ml的西尼罗病毒RNA,前者的敏感性比后者高100倍。而对其他黄病毒科成员的检测均为阴性,表明该方法是特异的。采用这种实时RT-PCR法可从西尼罗病毒感染小鼠的血液、脾脏、肾脏和脑组织标本中检测出病毒核酸,且在感染后第2天最先自血液和脾脏中检测到病毒,在感染后1周的脑组织中的病毒滴度最高。结论本研究建立的实时RT-PCR法具有很高的敏感性和特异性,因此该方法可用于西尼罗病毒疾病的早期监测和流行病学研究。

Detection of West Nile virus using real-time PCR assay

Objective To establish a fast, sensitive and specific real-time RT-PCR assay for early detection of West Nile virus(WNV) infection. Methods Titers of WNV in infected Vero cell supernatant were determined by plaque assay, then RNA of WNV genome was amplified by conventional and real-time RT-PCR assays. The sensitivities and specificities of two assays were compared. Results 0.02 PFU/ml and 2 PFU/ml of WNV RNA were detected by real-time and conventional RT-PCR assay, respectively. Sensitivity of the former was 100-fold greater than those of the latter. Results of the detection of other members of Flavivridae were negative, which indicated this assay was specific for WNV. In addition, WNV RNA could be detected from blood,spleen,kidney and brain samples of infected mice using the real-time RT-PCR assay, and the virus was detected in blood and spleen samples 2 days after infection, but virus titer reached the peak in brain one week after infection. Conclusion The real-time RT-PCR assay established in this study was highly sensitive and specific, and so can be used for early surveillance and epidemiology investigation of WNV infection.