多重荧光定量PCR甄别3种霍乱弧菌相关毒素基因

目的为了判定霍乱弧菌是否携带相关毒力因子,研发一种快速、准确、特异检测霍乱弧菌3种毒素基因的多重荧光定量PCR方法。方法针对霍乱弧菌ctxA、ace、zot基因设计引物和探针,建立PCR体系,对70株霍乱弧菌分离株进行鉴定,采用直接测序方法对鉴定结果进行验证。结果建立的多重实时荧光定量PCR方法可同时准确、特异地鉴定霍乱弧菌菌体携带的3种毒素基因,不携带毒素基因的菌株均未出现阳性检测结果。菌体检测的最低检出限度ctxA基因：102 CFU/mL,ace基因和zot基因：10CFU/mL。构建了3种毒素相关基因阳性质粒,ace基因和zot基因的最低检出限度为10copies/μL,ctxA基因的最低检出限度为102 copies/μL,定量检测批间和批内的变异系数均小于5%;对70株霍乱弧菌分离株进行评价,结果显示其中40株（57.2%）携带ctxA毒素基因、31株（44.3%）携带ace毒素基因、46株（65.7%）携带zot毒素基因,通过测序方法验证,符合率达到100%。结论本文建立的多重实时荧光定量PCR方法操作简便、特异性好、灵敏度高,为霍乱弧菌的现场流行病学调查和疫情监测提供了快速、可靠的鉴定工具。

Discrimination on three related toxin genes of Vibrio Cholerae using by multiplex real time PCR

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.