应用Taq Man荧光定量RT-PCR快速检测肠道病毒的研究

Application of TaqMan based real time fluorescent quantitative RT-PCR assay in rapid detection of enteroviruses

目的建立一种快速、灵敏、特异的荧光定量RT-PCR检测肠道病毒的方法。方法根据GenBank中肠道病毒5′UTR序列,应用生物软件在保守区设计与筛选特异引物和TaqMan探针,对荧光定量RT-PCR反应体系与条件进行优化,验证方法的特异性、敏感性和重复性。并通过对急性临床样本的检测,评价该方法的实际应用价值。结果该方法对肠道病毒包括脊髓灰质炎病毒Ⅰ、Ⅱ、Ⅲ型及柯萨奇病毒和埃可病毒等的检测有高度特异性,甲肝病毒、乙脑病毒、登革病毒、腺病毒、单纯疱疹病毒、诺如病毒等均呈阴性。该方法的检测下限达10-1TCID50/100μl。12份急性结膜炎病例结膜拭子标本中7份肠道病毒核酸阳性,普通RT-PCR方法5份阳性。结论TaqMan荧光定量RT-PCR方法快速、敏感、特异,适于肠道病毒的快速检测。

Objective To establish a rapid, sensitive and specific TaqMan RT-PCR method to detect the enteroviruses. Methods Specific primers and TaqMan probes were designed based on the alignment results of the 5＇ untranslated region （5＇-UTR） of the enteroviruses. The reaction systems and conditions were optimized. The sensitivity, specificity and repeatability of the method were validated. And the practical application of the method for the clinical samples was evaluated Results TaqMan RT-PCR was specific to detect the polioviruses type Ⅰ , Ⅱ and Ⅲ , the cosaxieviruses and eochviruses. But no reaction was observed with the hepatitis A virus, Japanese encephalitis virus, Dengue virus, adenovirus, Herpes simplex virus and norovirus. The detection limit was 10-1 TCID50/100 μl. Among the 12 conjunctiva swab samples from the acute conjunctivitis patients, there were 7 swabs positive detected by TaqMan RT-PCR, while there were 5 swabs positive by general RT-PCR. Conclusion TaqMan RT-PCR is a rapid, sensitive and specific method and could be used to detect the enteroviruses in laboratory.