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目的运用降落聚合酶链反应(touchdown PCR,TD-PCR)技术快速从腹泻、手足口病人粪便标本中同时检测诺如病毒(Norovius,NV)、肠道病毒71型(Enterovirus 71 EV71)和柯萨奇病毒A组16型(Coxsachie virus,CoxA16),并评价该技术用于检测这3种病毒的效能和敏感性。结论根据touchdown PCR原理设计TD-PCR程序,试验选择最佳反应条件,与普通PCR扩增效果进行评价。用10倍稀释检验TD-PCR方法的灵敏度,并用轮状病毒、札幌病毒、星状病毒、伤寒杆菌、小肠结肠炎耶尔森菌、香港海鸥形菌检测该方法的特异性。结果 TD-PCR能在一个反应条件下同时检测NV、EV71和CoxA16,扩增片段条带清晰,检测效果明显优于普通PCR,测序证实了3组片段的特异性,3种病毒cDNA的最低检测浓度分别为4.775μg/ml、2.360μg/ml、43.273μg/ml。检测该方法特异性时,其他病原体未见特异性条带扩增。结论成功建立的TD-PCR方法可同时检测腹泻和手足口病人粪便中NV、EV71和CoxA163种病毒,为快速检测相关病原体、科学防治腹泻和手足口病疫情暴发提供了可靠手段。
Objective To detect Norovius (NV), Enterovirus 71 EV71 and Coxa in diarrhea and hand-foot-mouth patients by touchdown PCR (TD-PCR) Coxsachie virus (CoxA16), and evaluated the efficacy of the technique for detecting the efficacy and sensitivity of the three viruses. Conclusion The TD-PCR program was designed according to the touchdown PCR principle. The optimum reaction conditions were selected for the experiment, and the PCR amplification was evaluated. The sensitivity of the TD-PCR method was examined by 10-fold dilution and the specificity of the method was examined using rotavirus, Sapovirus, Astrovirus, Salmonella typhi, Yersinia enterocolitica, and Haemonchus guinea. Results TD-PCR could simultaneously detect NV, EV71 and CoxA16 under one reaction condition. The bands of the amplified fragments were clear and the detection results were obviously better than those of the normal PCR. The sequencing confirmed the specificity of the three groups of fragments and the lowest detection of the three virus cDNAs The concentrations were 4.775μg / ml, 2.360μg / ml and 43.273μg / ml respectively. No specific bands were detected in other pathogens when tested for specificity. Conclusion The established TD-PCR method can detect NV, EV71 and CoxA163 viruses in diarrhea and hand-foot-mouth patients simultaneously, which provides a reliable method for rapid detection of related pathogens, scientific prevention and treatment of diarrhea and hand-foot-mouth disease outbreaks.