微孔杂交检测登革病毒及其分型方法的建立  

Detection and typing of dengue virus using polymerase chain reaction and microwell plate hybridization

作　　者：任瑞文[1] 徐晓立[1] 李建军[2] 方美玉[1] 刘建伟[1] 马安德 

机构地区：[1]广州军区疾病预防控制中心,广东广州510507 [2]南方医科大学分析测试中心,广东广州510515

出　　处：《南方医科大学学报》2006年第9期1356-1358,1362,共4页Journal of Southern Medical University

摘　　要：目的建立特异、敏感、实用的登革病毒检测及分型方法。方法分析登革1￣4型病毒基因组序列,分别设计针对登革病毒1￣4型的特异性包被探针,扩增、克隆测序后包被聚乙烯微孔板。PCR上游引物5'端标记生物素,对待检样品进行扩增后用作检测探针进行微孔杂交(PCR-ELISA)反应,并通过设定不同的包被DNA浓度、包被时间、杂交温度、杂交时间,对PCR-ELISA反应进行优化。结果建立了快速、特异、敏感的登革病毒快速检测及分型方法,试验结果直观,阳性标本吸光度值在0.5以上,阴性结果吸光度值在0.1以下,S/N值在10以上。结论所建立的方法可用作登革病毒感染的早期诊断及分型。Objective To establish a specific, sensitive and practicable method for detection and typing of dengue virus. Methods Based on the genomic sequence analysis of dengue virus types 1-4, 4 pairs of primers were designed. The specific capture probes of dengue virus types 1-4 were amplified using RT-PCR, cloned and sequenced before using them for precoating the microwell plate. The samples were amplified using biotin-labeled forward primer and reverse primer, and microwell plate hybridization was carried out for detection and typing of dengue virus types 1-4. Results The absorbance of the positive samples were higher than 0.5, while the average absorbance of the negative samples was lower than 0.1, with the S/N higher than 10. Conclusion The method of PCR-ELISA we established for early detection and typing of all 4dengue viruses seretypes.

关 键 词：登革热病毒 聚合酶链反应 酶联免疫吸附测定 杂交 

分 类 号：R450[医药卫生—治疗学]