常见腹泻病毒多重荧光逆转录聚合酶链反应检测方法的建立及临床应用

Construction and clinical application of a multiplex real-time reverse-transcript PCR for the detection of common diarrhea virus

目的建立能同时检测星状病毒、轮状病毒、肠道腺病毒和诺如病毒的多重荧光逆转录聚合酶链反应(RT-PCR)方法。方法根据上述4种病毒基因组保守序列设计引物和探针,建立并分析多重荧光RT-PCR的特异性、敏感性、灵敏度;以所建立方法对256例病毒性腹泻患者粪便标本进行检测,同时以基因测序进行验证。结果所建立的腹泻病毒多重荧光RT-PCR检测方法对具有星状病毒、轮状病毒、肠道腺病毒、诺如病毒特异性,灵敏度可达500 copy/mL。256例粪便标本中,星状病毒、轮状病毒、肠道腺病毒和诺如病毒的检出率分别为3.13%、42.97%、9.38%和10.16%,与基因测序比对结果相符。结论成功构建可用于常见腹泻病毒检测的多重荧光RT-PCR检测方法,该法具有快速、灵敏、特异等优点,可用于临床病原诊断。

Objective To construction a multiplex real-time reverse-transcript polymerase chain reaction（RT-PCR） to detect Astrovirus,Rotavirus,Enteric Adenovirus and Norovirus.Methods Primers and probes were designed according to the conserved genome sequence of 4 virus mentioned above.A multiplex real-time RT-PCR was constructed,and the specificity and sensitivity of it were evaluated.Fecal samples from 256 cases of patients with viral diarrhea were detected by constructed method and the results were compared with gene sequencing.Results The constructed multiplex real-time RT-PCR was specific for Astrovirus,Rotavirus,Enteric Adenovirus and Norovirus,with sensitivity about 500 copy/mL.In 256 fecal samples,the detection rates of Astrovirus,Rotavirus,Enteric Adenovirus and Norovirus were 3.13%,42.97%,9.38% and 10.16%,respectively,which were in accord with gene sequencing.Conclusion A new multiplex real-time RT-PCR for the detection of common diarrhea virus was successfully constructed,with the advantages such as rapid detecting,sensitive and specific,and could be used for the clinical pathogen diagnosis.