RT-PCR与SYBR GreenⅠ实时RT-PCR方法检测乙型脑炎病毒的比较

Comparison of RT-PCR and SYBR Green Ⅰ real time RT-PCR assays for detecting Japanese encephalitis virus

目的:比较RT-PCR和SYBR GreenⅠ实时RT-PCR方法检测乙型脑炎病毒(JEV)的敏感性、特异性和时效性。方法:采用RT-PCR和SYBR GreenⅠ实时RT-PCR方法对连续10倍稀释的JEV核酸样品平行检测,比较两种方法的敏感性。同时以登革2型病毒、诺如病毒和狂犬病毒核酸进行两种方法的特异性测试。结果:SYBR GreenⅠ实时RT-PCR方法可检测出40copies/μL的核酸分子水平,与普通RT-PCR方法相比,敏感度提高了10倍,检测时间节省2h。两种检测方法在检测JEV方面,与登革2型病毒、诺如病毒和狂犬病毒核酸均无交叉反应。结论:与普通RT-PCR相比,SYBR GreenⅠ实时RT-PCR是一种更敏感、快速检测JEV的方法。

Objective To compare the sensitivity, specificity and chronergy of detecting Japanese encephalitis virus （JEV） by RT-PCR and SYBR Green Ⅰ real-time RT-PCR assays. Methods 10-fold serial dilutions of the JEV RNA were used for detection of sensitivity of the RT-PCR and SYBR Green Ⅰ real-time RT-PCR assay. While dengue type 2 virus, norovirus, and rabies virus nucleic acid were tested to verify the specificity of the two assays. Results Sensitivity of SYBR Green Ⅰ real-time RT-PCR assay was 40 copies/μL which was 10 fold more sensitive than that of conventional RT-PCR assay. There was no crossing-reaction with dengue type 2 virus, norovirus and rabies virus. Compared with conventional RT-PCR, the SYBR Green Ⅰ real-time RT-PCR had been saving about two hours. Conclusion Compared to conventional RT-PCR, the SYBR Green Ⅰ real-time RT -PCR is a more sensitive and rapid assay for detection of JEV.