TaqMan荧光定量RT-PCR快速检测登革1、2型病毒

TaqMan-Based Real-Time PCR Assay for Quick Detection of Dengue 1,2 Serotype Virus

目的建立特异、灵敏、快速的荧光定量RT-PCR方法用于检测登革1、2型病毒核酸,并初步应用于登革热的临床标本检测。方法根据GenBank登录的登革热毒株序列,应用生物学软件进行序列比对,分别在登革病毒1型的NS5基因和2型的C基因的保守区设计特异性引物和TaqMan探针。对荧光定量RT-PCR反应条件进行优化,验证该方法的特异性、灵敏度和稳定性。同时对疑似登革热病例血清标本进行检测。结果该方法对登革1、2型病毒的检测有高度的特异性,与登革病毒的4个血清型之间以及流行性乙型脑炎病毒、肾综合征出血热病毒等均无交叉反应,检测的灵敏度均达0.1TCID50,可从疑似登革热病例血清标本中直接检测登革病毒核酸,从病毒核酸提取至完成检测仅需3h左右。结论本研究建立的TaqMan荧光定量RT-PCR是一种快速检测登革1、2型病毒特异、敏感的方法,适用于临床早期诊断。

Objective To establish a TaqMan-based real-time PCR assay for detection of dengue 1,2 serotype virus, and the constructed method was primarily used to clinical samples test for dengue. Methods The gene sequences of dengue virus downloaded from the GenBank was aligned using the biologic software and the specific primers and probes were designed in the conserved region of the NS5 for 1 serotype and C gene for serotype. The primers, probes and the reactive condition were optimized to improve the sensitivity and specificity of the assay. The clinical specimens collected from the patients infected with dengue fever were detected by this assay. Results The specificity of the assay was high and there were no cross reactions with Japanese encephalitis virus（JEV）, haemorrhagic fever with renal syndrome（HFRS） and other serotype dengue virus. The sensitivity of the assay was 0.1 TCID50 and the viral RNA was directly detected with the clinical specimens by this assay. It took all about three hours to extract viral RNA and the real-time PCR. Conclusion This TaqMan-based real-time PCR assay was a quick, sensitive and specific method for molecular diagnosis of dengue 1,2 serotype virus.