人星状病毒核酸检测方法的建立及应用

Establishment and Application of Human Stellavirus Nucleic Acid Detection Method

目的:建立人星状病毒的实时荧光定量RT-qPCR检测方法,检测胃肠道感染患儿标本中的HAstV。方法:设计HAstV ORF2基因的引物和Taqman探针,扩增ORF2基因片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品,建立RT-qPCR检测方法,进行线性、准确性、特异性试验,并检测195份临床标本。结果:建立的RT-qPCR方法线性范围良好,准确性100%,对临床其他消化道病毒特异性好,195份临床标本中HAstV检测阳性15份,阳性率为7.7%。结论:成功建立了HAstV实时荧光定量RT-qPCR检测方法,并应用于临床标本的检测,为人星状病毒感染的临床治疗打下坚实的基础。

objective:to establish a real-time fluorescent quantitative RT-qPCR method for the detection of human stellavirus,and to detect HAstV in the samples of children with gastrointestinal tract infection.Methods:the primers of HAstV ORF2 gene and the Taqman probe were designed,the ORF2 gene fragment was amplified and cloned into the pGEM-T Easy vector,the plasmid standard was constructed,the RT-qPCR detection method was established,the linear,accuracy and specificity tests were conducted,and 195 clinical samples were tested.Results:the established RT-qPCR method had good linear range,100%accuracy and specificity for other clinical digestive tract viruses.Among the 195 clinical samples,15 were positive for HAstV,with a positive rate of 7.7%.Conclusion:the method of HAstV real-time fluorescence quantitative RT-qPCR has been successfully established and applied to the detection of clinical specimens,which lays a solid foundation for the clinical treatment of human stavirus infection.