摘要
目的建立能同时检测轮状病毒A群和星状病毒双重实时荧光定量反转录-聚合酶链反应(rRT-PCR)方法。方法根据上述2种病毒基因组保守序列设计引物和探针,建立并分析多重rRT-PCR的重复性、特异性、敏感性;以所建立方法对128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。结果所建立的双重rRT-PCR检测方法对轮状病毒A群和星状病毒具有很好的特异性和重复性,轮状病毒A群灵敏度达到每个反应101拷贝,星状病毒灵敏度达到每个反应102拷贝。128例粪便标本中,轮状病毒A群和星状病毒的检出率分别为18.0%和3.1%。通过基因测序比对结果相符。结论构建可用于轮状病毒A群及星状病毒的双重rRT-PCR方法快速、特异且灵敏,可用于临床病原诊断和流行病学调查。
Objective To establish and evaluate a single-tube multiplex real time RT-PCR assay for detecting rotavims group A and astrovirus simultaneously. Methods The primers and probes were designed according to the conserved genome sequence of the 2 viruses mentioned above. A multiplex real-time RT-PCR assay was established. The stability, specificity and sensitivity of the assay were evaluated. The fecal samples from 128 patients with viral diarrhea were detected by the established assay and the results were compared through gene sequencing. Results The established multiplex real-time RT-PCR assay was specific for both rotavirus group A and astrovirus. The stability test showed the co-efficient variables was all less than 2. 0% in 2 different samples. The detection limit of this assay was 101 copies/pal for rotavirus group A and 102 copies/μl for astrovirus in one reaction. Among the 128 clinical samples detected, 3.1% were astrovirus RNA positive and 18.0% were rotavirus group A RNA positive respectively. The positive samples were verified by sequencing. Conclusion The single-tube multiplex RT-real time PCR assay, established in this study for detecting and identifying rotavirus group A and astrovirus, is rapid, specific and sensitive and can be used in clinical etiological diagnosis and epidemiological investigation
出处
《疾病监测》
CAS
2014年第12期983-986,共4页
Disease Surveillance
