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目的建立一种单管巢式多重RT-PCR方法,以用于临床对于不同型别登革病毒混合感染的检测并分型。方法将1~4型登革病毒RNA混合,首先用1~4型病毒通用外引物进行一步法RT-PCR,然后以混合病毒RT-PCR产物为模板,在同一反应管内同时加入4对(5条)型特异性引物进行巢式多重PCR,结果用EB染色的3%琼脂糖凝胶电泳观察;并将其检测敏感性和特异性与该方法的单一病毒模板RNA扩增进行比较。结果经反应条件的优化,混合病毒模板单管巢式多重PCR可以在同一反应管内同时扩增出4条病毒特异性目的条带,分别为482、119、290、389 bp。其敏感性和特异性与单一病毒RNA的扩增相当,对模板RNA检测的敏感性可以达到66.068 copies/μl。结论该方法简便、快速、敏感、特异,可以根据扩增目的片段的大小进行诊断和分型,对于临床登革病毒混合感染病例的诊断和快速分型有应用价值。
Objective To establish a single-tube nested multiplex RT-PCR method for the clinical detection and typing of different types of dengue virus mixed infection. Methods 1 to 4 dengue virus RNAs were mixed. First, one-step RT-PCR was carried out with universal primers 1 to 4, and then mixed virus RT-PCR products were used as templates. In the same reaction tube, 4 pairs of 5 Strip) specific primers were used for nested multiplex PCR and the results were observed by EB stained 3% agarose gel electrophoresis; and the detection sensitivity and specificity were compared with that of the single viral template RNA of this method. Results After optimization of the reaction conditions, single-tube nested multiplex PCR of mixed-virus template could amplify four virus-specific bands in the same reaction tube simultaneously, which were 482,119,290,389 bp, respectively. Its sensitivity and specificity are comparable to that of a single viral RNA, and its sensitivity to template RNA detection can reach 66.068 copies / μl. Conclusion The method is simple, rapid, sensitive and specific, and it can be used for diagnosis and typing of the amplified fragments according to the size of the amplified fragments. It is of value in the diagnosis and rapid typing of clinical mixed dengue virus infection.