摘要
为建立一种基于TaqMan探针法定量检测霍乱弧菌的三重荧光定量PCR方法,以霍乱弧菌种特异性基因ompW和毒力基因ctx、hly为靶基因,分别设计特异性引物及相应的TaqMan探针。在ompW、ctx、hly探针的5’端分别标记CY5、FAM、HEX荧光报告基团,3’端均标记BHQ1荧光淬灭基团。结果显示,本试验建立的三重荧光定量PCR方法与其他菌株无交叉反应,其最低检出限达到了10 CFU/m L,重复性试验中每组变异系数均小于1.4%;检测人工染菌的虾肉和贝类样品时,最低检出限也达到1×102CFU/m L。结果表明,本实验室建立的三重荧光PCR检测方法具有灵敏度高、特异性强和重复性好的特点,是高通量检测致病性霍乱弧菌的有效手段。
To establish a triple real-time PCR assay for quantitative detection of Vibrio cholera based on Taq Man probe,species-specific gene omp W and the virulence genes ctx,hly of V.cholerae were used as target genes to design specific primers and Taq Man probes.The 5′-ends of probes for omp W,ctx and hly were individually labeled with CY5,FAM and HEX and the 3′-ends were all labeled with quencher BHQ1.In result,the specificity of the assay has no cross-reaction with other pathogens,the minimum detection limit is 10 CFU/m L,and that the variation coefficient of repeated experiments is less than1.4%.Triple real-time PCR method was further used to detect shrimp and shellfish samples contaminated by V.cholerae,and the minimum detection limit reached to 1×10~2 CFU/m L.The above-mentioned results showed that this newly established triple real-time PCR assay is cost-effective,specific,sensitive and capable of high-throughput detection of pathogenic V.cholerae.
作者
万莹
陈永军
任亚玲
王亚磊
王权
蒋蔚
WAN Ying;CHEN Yong-jun;REN Ya-ling;WANG Ya-lei;WANG Quan;JIANG Wei(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2019年第9期1143-1151,共9页
Chinese Veterinary Science
基金
上海市科学技术委员会科研计划项目（17140900400）
国家重点研发计划项目（2017YFC1200201）
