| 摘要: |
| 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。 |
| 关键词: 登革病毒 混合感染 单管巢式多重聚合酶链反应 |
| DOI：10.3724/SP.J.1008.2010.0152 |
| 投稿时间：2009-03-28修订日期：2010-01-20 |
| 基金项目:第43批中国博士后科学基金(20080431367)，军队“十一五”科技攻关计划课题基金(06G65)，上海市自然科学基金(07ZR14141). |
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| Rapid detection and typing of Dengue virus by single-tube nested multiplex-PCR |
| LI Shu-hua1 , FANG Mei-yu2, LIU Shi-jian1, CHANG Wen-jun1 , WANG Shan-shan2, CAO Guang-wen1* |
| (1. Department of Epidemiology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China;2. Department of Infectious Diseases, Center of Disease Control and Prevention of PLA Guangzhou Military Area Command, Guangzhou 510000, China) |
| Abstract: |
| ObjectiveTo establish a single-tube nested multiplex-PCR assay for rapid detection and typing of Dengue viruses for multiple infections with different serotypes. MethodsA pair of outer universal primers designed for all the four Dengue virus serotypes were used to amplify the mixed RNA of 1-4 dengue viral serotypes by one-step RT-PCR，and the products were used as template for nested multiplex PCR using four pairs of serotype-specific primers in the same reaction tube. The sensitivity and specificity of single-tube nested multiplex PCR assay amplifying from the mixed 1-4 serotype dengue viral RNA were subsequently compared with those amplifying from the single serotypes dengue viral RNA. ResultsBy optimizing the reaction condition，four specific fragments (482,119,290,and 389 bp) were successfully amplified from the mixed RNA of 1-4 serotypes dengue viruses in single tube by single-tube nested multiple-PCR. Its sensitivity and specificity amplifying from the mixed RNA of 1-4 serotypes dengue viruses were similar to those amplifying from the single serotype dengue viral RNA. The detection limit of nested multiple-PCR was 66.068 copies/μl. ConclusionSingle-tube nested multiple-PCR method is simple,rapid,sensitive, and specific for detecting and typing dengue viruses,and it is valuable for detecting and typing of the clinical multiple infections. |
| Key words: Dengue virus multiple-infection single-tube nested multiple-polymerase chain reaction |
