TD-PCR技术用于肠道病毒71的检测  

Touchdown PCR Method for Detection of Enterovirus71

作　　者：高璐璐[1,2] 陈清[1] 郭江[1] 陈志永[1] 俞守义[1] 

机构地区：[1]南方医科大学流行病学系,广州510515 [2]广州军区空军后勤部,广州510620

出　　处：《热带医学杂志》2009年第12期1377-1379,1384,共4页Journal of Tropical Medicine

摘　　要：目的评价降落聚合酶链反应(touchdown PCR,TD-PCR)检测肠道病毒71型(Enterovirus71,EV71)的效能和敏感性。方法根据touchdown原理设计TD-PCR程序,试验选择PCR最佳反应条件,分别与普通PCR及TD-PCR、普通Taq酶与热启动酶扩增效果进行比较,并利用10倍稀释法检验TD-PCR方法的灵敏度。琼脂糖凝胶电泳验证扩增产物,纯化后DNA产物测序验证该方法的特异性。结果 TD-PCR的扩增片段条带清晰,检测效果明显优于普通PCR,使用普通Taq酶的TD-PCR比使用热启动酶有更优的性价比,测序证实了此组片段的特异性,cDNA的最低检测浓度为1.031μg/mL。结论成功建立TD-PCR检测EV71的方法,使用普通Taq酶获得理想的检测结果,为快速检测手足口病的病原体提供了可靠的手段。Objective To evaluate the efficiency and sensitivity of touchdown PCR（TD-PCR） for Enterovirus 71. Methods The optimized procedures of decreasing annealing temperature in a certain scale were designed for optimal PCR conditions. The results of regular PCR and TD-PCR were compared, meanwhile the results of using regular Taq DNA polymerase and Hot Start Taq were compared. The sensitivity of TD-PCR was determined by tenfold serial diluted eDNA. Then amplified products were analyzed by agarose gel electrophoresis and sequencing. Results TD- PCR procedure whose annealing temperature was set from 55℃to 50℃for optimized PCR conditions was designed. The data suggested that TD-PCR is more effective than conventional PCR, and more economic than PCR using Hot Start Taq. The lowest detection level of TD PCR was 1.031 μg/mL. Conclusion Successfully establishing touchdown PCR technique and providing an quick and reliable way to detect EV71, which is a novel TD-PCR using regular polymerase was established for detecting EV71 quickly and stably.

关 键 词：降落聚合酶链反应 肠道病毒71型 检测 

分 类 号：R446.6[医药卫生—诊断学]