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 (ctxA), accessory cholera enterotoxin 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 10cfu/mL for ace and zot gene in pure culture. The standard plasmids according to each toxin gene were constructed, and consequently the detection limit of ace gene and zot gene was 10copies/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.
收稿日期: 2011-06-17
出版日期: 2011-12-20
基金资助:艾滋病和病毒性肝炎等重大传染病防治科技重大专项;浙江省卫生厅课题
通讯作者:
金大智
E-mail: dzjin@cdc.zj.cn
引用本文:
金大智. 多重荧光定量PCR甄别三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.
dazhi Jin. Discrimination on three related toxin genes of Vibrio Cholerae using by multiplex real time PCR. Chinese Journal of Zoonoses, 2011, 27(12): 1065-1070.