A组轮状病毒极速实时荧光RT-PCR检测方法的建立及临床评价

Establishment and clinical evaluation of a rapid real-time RT-PCR for detection of rotavirus A

目的建立一种A组轮状病毒(rotavirus A,RVA)极速实时荧光RT-PCR检测方法。方法根据RVA基因组中非结构蛋白(non-structural rotavirus protein 3,NSP3)基因保守区序列设计一对引物和一条荧光探针,建立RVA极速实时荧光RT-PCR技术检测方法并对其进行灵敏度、特异性、精密度分析,最后采用200例临床粪便样本同时进行该方法检测和测序结果对比分析,以评价本方法的临床应用性能。结果本方法检测RVA的灵敏度达到2.00×102PFU/m L;对种属相近或引起相似症状的其他种属病毒无交叉反应;不同浓度样本的Ct值的变异系数在0.82%～2.28%之间。200例临床样本的研究结果显示本方法与测序方法检测结果的总符合率达到98.5%。结论本研究建立的方法检测RVA具有灵敏、特异、精密度高、快速简便的特点,并与测序方法具有很好的符合率,在病毒性腹泻的快速诊断和疫情监测方面具有很好的应用前景。

Objective To establish a rapid and early detection method of rotavirus A(RVA)by real-time reverse-transcription polymerase chain reaction(RT-PCR).Methods A pair of primers and a fluorescence probe based on conservative sequences of non-structural rotavirus protein 3(NSP3)in RVA genomes were designed to establish a RVA detection method by real-time RT-PCR,and then its sensitivity,specificity and precision were analyzed.A total of 200 cases from clinical stool samples were tested simultaneously and sequencing to evaluate the clinical performance of this method.Results The sensitivity of the method for detecting RVA reached 2.00×102 PFU/m.There was no cross-reactivity to other species viruses with similar species or similar symptoms.The coefficient of variation of Ct values of different concentrations of samples ranged from 0.82%to 2.28%.The results from 200 clinical samples showed that the total coincidence rate between the method and the sequencing method reached 98.5%.Conclusion The method established in this study has the characteristics of sensitive,specific,high precision,fast and simple,and has a good coincidence with sequencing methods.It has a good application prospect in the rapid diagnosis and epidemic monitoring of viral diarrhea.