EVAGreen实时荧光定量PCR检测类志贺邻单胞菌的方法建立  

Establishment of a real-time PCR with EVAGreen dye for the detection of Plesiomonasshigelloides

作　　者：陈智瑾[1] 廖虹瑜[1] 杨晶艳[1] 曹霞[1] 胡鹏威[1] 叶倩[1] 裴晓方[1] 

机构地区：[1]四川大学华西公共卫生学院医学检验教研室,四川成都610041

出　　处：《现代预防医学》2012年第23期6234-6237,共4页Modern Preventive Medicine

基　　金：国家科技重大专项"传染病监测技术平台"项目(2009ZX10004-212)资助;欧盟食品安全MoniQA项目(FOOD-CT-2006-036337)资助

摘　　要：目的建立实时荧光定量PCR快速检测类志贺邻单胞菌的方法。方法以类志贺邻单胞菌23SrRNA基因为目标设计引物,使用EVAGreen染料建立实时荧光定量PCR方法并优化反应条件,构建重组质粒作为标准DNA,建立标准曲线;评估该方法的灵敏度、重复性和特异性,并将所建方法用于粪便模拟样品的检测,与常规培养法比较。结果在反应体系为10μl,模板为1μl的条件下,该方法可测的最低拷贝数为2.18×101拷贝/体系;重组质粒标准建立的标准曲线具有较大的线性范围(108～102copies/μl);批间差异小于5%,批内差异小于3%;特异性试验中类志贺邻单胞菌检测结果均为阳性,其他常见肠道致病菌、常见弧菌以及常见正常肠道菌均为阴性;粪便模拟样品前增菌处理后检测灵敏度为25cfu/10ml增菌液,检测结果和传统培养法相符。结论成功建立EVAGreen实时荧光定量PCR方法,该方法的建立为类志贺邻单胞菌的检测提供了一种简便、快捷的途径,并可对样品中类志贺邻单胞菌进行快速定量检测。OBJECTIVE To establish a method of real-time quantitative polymerase chain reaction with EVAGreen dye for detection of Plesiomonasshigelloides. METHODS Two primers targeted against part of the 23S rRNA gene of P. shigelloides were designed. After optimization of reaction conditions of real-time PCR, the recombinant plasmids were constructed for the establishment of standard curve. Then, the sensitivity, specificity and reproducibility were evaluated. Finally, the established method was conducted to 10 fecal samples., which was compared with traditional culture method. RESULTS In the conditions of reaction system for 10 μl and template for 1 μl, the detection limit was 2.18×10^1/ μl.Dynamic range was 108 copies / μl-102 copies / μl. Coefficients of variation intra-assay and inter-assay over the dynamic range were lower than 3% and 5% respectively. Plesiomonasshigelloides were found positive and non -Plesiomonasshigelloides were negative.After 16 h enrichment, Plesiomonasshigelloides was detected in fecal samples with the sensitivity of 25 cfu per sample, and the results were in agreement with the traditional culture method. CONCLUSION Results from our study indicate that this method might serves as a tool for a rapid, quantitative and sensitive identification of P. shigelloides, and it can be applied in sample testing.

关 键 词：类志贺邻单胞菌 实时荧光定量PCR EVAGreen 23SrRNA 检测 

分 类 号：R115[医药卫生—公共卫生与预防医学]