实时荧光定量聚合酶链式反应快速检测4种食源性致病弧菌

Rapid determination of 4 kinds of foodborne pathogenic species of Vibrio by quantitative real-time polymerase chain reaction

中文关键词:  副溶血性弧菌  霍乱弧菌  拟态弧菌  创伤弧菌  荧光定量聚合酶链式反应

英文关键词:Vibrio parahaemolyticus  Vibrio cholera  Vibrio mimicry  Vibrio vulnificus  fuorogenic quantitative polymerase chain reaction

中文摘要:

      目的 建立和优化4种食源性致病弧菌的实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)快速检测方法。方法 基于副溶血性弧菌的toxR基因(GenBank ID: MH047287.1)、霍乱弧菌的ompW基因(GenBank ID: MF100046.1)、拟态弧菌的toxR基因(GenBank ID: EF693743)和创伤弧菌的vvhA基因(GenBank ID: KC821520.1)设计常规PCR引物和qPCR特异性引物和探针。分别建立副溶血性弧菌、霍乱弧菌、拟态弧菌和创伤弧菌4种食源性致病弧菌的单重qPCR检测体系, 根据扩增曲线和Ct值对探针浓度进行优化, 设置探针浓度梯度。结果 4种弧菌的探针最适浓度均为0.1 μmol/L。对设计的4种弧菌的引物分别进行常规PCR的扩增, 其最低检出限均为1×105 copies/μL, 4种弧菌分别进行单重qPCR的最低检出限均为1×101 copies/μL, 扩增效率均接近100%。qPCR的灵敏度均高于常规PCR。特异性实验结果表明, 4种目的弧菌DNA扩增出特异性曲线, 其他的非目的基因未被扩增出扩增曲线。结论 该方法准确度和灵敏度高, 前处理简单快速, 适用于4种食源性致病弧菌的荧光定量PCR快速检测。

英文摘要:

      Objective To establish and optimize the rapid detection method of 4 kinds of foodborne pathogenic Vibrio by quantitative real-time polymerase chain reaction (qPCR). Methods The toxR gene of Vibrio parahaemolyticus (GenBank ID: MH047287.1), ompW gene of Vibrio cholerae (GenBank ID: MF100046.1) and toxR gene of Vibrio mimicry (GenBank ID: EF693743) and vvhA gene of Vibrio vulnificus (GenBank ID: KC821520.1) were used to design primers for conventional PCR and specific primers and probes for real-time fluorescence quantitative PCR. The detection system of single real-time fluorescence quantitative PCR for 4 kinds of foodborne pathogenic Vibrio species, including Vibrio parahaemolyticus, Vibrio cholerae, Vibrio mimicry and Vibrio vulnificus, was established. The probe concentration was optimized according to the amplification curve and Ct value, and the probe concentration gradient was set. Results The optimal probe concentration for 4 kinds of Vibrio species was 0.1 μmol/L. The minimum limit of detection of conventional PCR amplification with 4 kinds of real-time quantitative PCR primers was 1×105 copies/μL, and the minimum limit of detection of single qPCR for 4 kinds of Vibrio strains was 1×101 copies/μL, and the amplification efficiency was close to 100%. The experimental results showed that the sensitivity of qPCR was higher than that of conventional PCR. The results of the specificity test showed that only 4 kinds of target Vibrio DNA could be amplified with the amplification curve, and the other non-target genes could not be amplified without the amplification curve. Conclusion The method has high accuracy and sensitivity, simple and fast pre-treatment, and is suitable for 4 kinds of foodborne pathogenic Vibrio by fluorescence quantitative PCR.