Koutango病毒TaqMan反转录聚合酶链式反应方法的建立

Establishment of TaqMan RT-PCR for detection of Koutango virus

目的建立Koutango病毒(KOUTV)的实时荧光定量TaqMan反转录聚合酶链式反应(RT-PCR)检测方法。方法从GenBank中下载KOUTV基因序列,进行多序列比对分析,针对NS5基因中的高保守序列片段设计KOUTV的特异引物与探针,用4科9种病毒进行检测方法特异性验证,通过平行重复实验验证检测方法稳定性,利用体外转录的RNA标准品建立了KOUTV的NS5基因拷贝数的绝对定量分析模型。结果引物与探针工作特异性良好,同一样品重复检测循环阈值(Ct)的变异系数均小于1.5%,定量分析模型灵敏度达到1.0×10^2拷贝/反应。通过本研究建立的快速检测方法对新疆采集的112批,6328只蚊虫进行了测定,结果均为阴性。结论成功建立了KOUTV的高特异性、高敏感性的TaqMan RT-PCR检测方法,可应用于实验室和临床诊断。

Objective To establish a real-time quantitative TaqMan reverse transcription-polymerase chain reaction(RTPCR)assay for the rapid detection of Koutango virus(KOUTV)in mosquito borne arbovirus surveillance.Methods All gene sequences of KOUTV were downloaded from GenBank for multiple sequence alignment.Specific primers and probes were designed for the highly conserved region of NS5 gene.The specificity of established TaqMan RT-PCR assay was evaluated with other 9 viruses from 5 families,and the stability of the assay was verified by parallel repeated experiments.Moreover,an absolute quantitative analysis model for the NS5 gene copy number of KOUTV was set up with in vitro transcribed RNA standards.Results The established TaqMan RT-PCR assay has good specificity and sensitivity.No cross reaction with other arbovirus was observed and the sensitivity was 1.0×10^2 copies/reaction.The coefficients of variation of Ct values in repeated detection of same sample were all less than 1.5%.A total of 6328 mosquito samples in 112 batches collected in Xinjiang were tested with the established assay and the results showed KOUTV negative in all samples.Conclusion A high specificity and sensitivity TaqMan RT-PCR assay for KOUTV was successfully established.