多重聚合酶链式反应技术检测罗非鱼5种常见食源性致病菌

Determination of 5 kinds of common foodborne pathogens in Oreochromis niloticus by multiplex polymerase chain reaction technology

中文关键词:  罗非鱼  食源性致病菌  多重聚合酶链式反应  快速检测

英文关键词:Oreochromis niloticus  foodborne pathogens  multiplex polymerase chain reaction  rapid detection

中文摘要:

      目的 建立一种可同时检测无乳链球菌(Streptococcus agalactiae)、嗜水气单胞菌(Aeromonas hydrophila)、霍乱弧菌(Vibrio cholerae)、大肠杆菌(Escherichia coli)和沙门菌(Salmonella) 5种罗非鱼常见食源性致病菌的多重聚合酶链式反应(polymerase chain reaction, PCR)方法。方法 根据5种致病菌特异性基因片段设计并合成引物, 优化多重PCR体系条件, 并对多重PCR体系的特异性、灵敏度以及人工模拟样品进行检测。结果 建立的多重PCR方法可同时扩增5种目的菌株的特异性条带, 且不与非靶标细菌发生交叉反应。敏感性实验结果显示, 该方法对无乳链球菌、嗜水气单胞菌、沙门菌、霍乱弧菌、大肠杆菌纯培养物基因组DNA的检出限均为0.4 ng/μL。人工模拟样品检测结果显示, 该方法可以快速且准确地检测上述5种食源性致病菌, 且检出限可达到2×101 CFU/g。结论 本研究建立了一种可同时检测无乳链球菌、嗜水气单胞菌、霍乱弧菌、大肠杆菌和沙门菌5种罗非鱼常见食源性致病菌的多重PCR检测方法。该方法的建立为罗非鱼常见食源性致病菌的快速检测提供了重要的技术手段。

英文摘要:

      Objective To establish a method for simultaneous determination of 5 kinds of common foodborne pathogens in Oreochromis niloticus, including Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli and Salmonella by multiplex polymerase chain reaction (PCR). Methods Primers were designed and synthesized according to specific gene fragments of 5 kinds of pathogenic bacteria. The conditions of multiplex PCR system were optimized, and the specificity, sensitivity and artificial simulation samples of multiplex PCR system were detected. Results The established multiplex PCR method could simultaneously amplify the specific bands of 5 kinds of target strains without cross-reaction with non-target bacteria. The sensitivity test showed that the limit of detection of this multiplex PCR method was 0.4 ng/μL for the genomic DNA of pure cultures of Streptococcus agalactiae, Aeromonas hydrophila, Salmonella, Vibrio cholerae, Escherichia coli. The artificially simulated sample testing showed that this method could quickly and accurately detect the above 5 kinds of foodborne pathogens, and the limit of detection could reach 2×101 CFU/g for each pathogen. Conclusion A quintuple PCR method to identify 5 kinds of common foodborne pathogens in Oreochromis niloticus has been developed in this study. The establishment of this method provides an important technical means for the rapid detection of common foodborne pathogens in Oreochromis niloticus.