多重RT-PCR结合反向杂交技术检测多种流感病毒方法的研究  

The study of multiple RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses

作　　者：杨亮[1] 张晓梅[2] 张晓光[2] 马晶[4] 王敏[3] 温乐英[3] 王大燕[3] 白天[3] 舒跃龙[3] 钱永华[1] 曾毅[2] 

机构地区：[1]西北农林科技大学动物医学院,712100 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室 [3]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室 [4]北京工业大学生命科学与生物工程学院

出　　处：《中华实验和临床病毒学杂志》2010年第5期383-385,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：＂人禽流感阻断技术研究＂国家科技支撑计划（课题任务书编号：2006BAD06A15）

摘　　要：目的 建立一种基于多重RT-PCR结合反向斑点杂交技术的多种流感病毒核酸快速检测方法.方法 对5种亚型流感病毒的HA基因序列进行比对分析,选择合适的保守区域设计引物序列,并在扩增产物序列内部选择高变区设计探针序列.首先,使用生物素标记的引物扩增出同样带标记的PCR产物;然后,产物与膜上的探针杂交,通过生物素-亲和素反应,最后以斑点显色的方式来检测和鉴别不同亚型的流感病毒.实验中还通过摸索不同的引物浓度和探针浓度来优化反应条件.结果 成功建立了一种基于多重RT-PCR结合反向斑点杂交技术的检测方法,该方法能够成功检测和鉴别5种不同亚型的流感病毒,检测灵敏度比传统的RT-PCR方法高100～1000倍.结论 成功建立了一种可高通量快速检测多种流感病毒的多重RT-PCR结合反向斑点杂交技术的检测方法.Objective To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses. Methods Obtain the HA nucleotide sequences of seasonal influenza H1 N1, seasonal influenza H3N2, influenza H1 N1 and human avian influenza H5 N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance. Results Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.Conclusion Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.

关 键 词：聚合酶链反应 杂交 流感 

分 类 号：R440[医药卫生—诊断学]