中国人兽共患病学报2011，Vol.27Issue(12)：1065-1070,6.
多重荧光定量PCR甄别3种霍乱弧菌相关毒素基因
Discrimination on three related toxin genes of Vibrio Cholerae using bv multiplex real time PCR
摘要
Abstract
In order to assess Vibrio Cholerae which containing related toxin genes, a rapid, sensitive and specific assay based on multiplex real time PCR was developed in this study. The cholera toxin sub-unit A gene CctxA), accessory cholera en-terotoxin gene (ace) , and zonula occludens toxin gene (zot) of Vibrio Cholerae was chosen as targets, and then the primers and TaqMan probe were designed. Furthermore, multiplex real time PCR was applied to detect 70 Vibrio Cholerae isolated from samples, and PCR products were sequenced in order to affirm the results of multiplex real time PCR. The results showed that ctxA, ace and zot gene of Vibrio Cholerae were detected by using multiplex real time PCR accurately and quickly. When other bacteria containing no all three target toxin genes were detected, no positive results appeared. The sensitivity was 102cfu/mL for ctxA gene and lOcfu/mL for ace and zot gene in pure culture. The standard plasmids according to each toxin gene were con-structed, and consequently the detection limit of ace gene and zot gene was lOcopies/μL and the detection limit of ctxA gene was 102copies/μL. The coefficient of variation of intra-assay and inter-assay was less than 5. 0%. When this assay was applied directly to identify 70 Vibrio Cholerae, the results showed that 40 strains (57. 2%) were positive to ctxA gene, 31 strains (44. 3%) were positive to ace gene, and 46 strains (65.7%) were positive to zot gene. The results above were the same to the results obtained from the sequencing assays. The coincidence was 100%. It is demonstrated that multiplex real time PCR is a simple, accurate and feasible assay for discriminating three related toxin genes of Vibrio Cholerae. The assay described here provided a reliable tool for epidemiologic survey and epidemic monitoring.关键词
多重实时荧光定量PCR/霍乱弧菌/毒素Key words
multiplex real time PCR/Vibrio Cholerae/ toxin分类
医药卫生引用本文复制引用
金大智,张政,罗芸,叶菊莲,程苏云,吴方,金郁..多重荧光定量PCR甄别3种霍乱弧菌相关毒素基因[J].中国人兽共患病学报,2011,27(12):1065-1070,6.基金项目
"艾滋病和病毒性肝炎等重大传染病防治科技重大专项""传染病检测技术研究-传染病病原体诊断和组合检测技术"课题(2008ZX10004-002)和"霍乱弧菌核酸快速甄别方法研究及分子分型数据库的建立"课题(2010KYB110)联合资助 （2008ZX10004-002）
