TaqMan探针双重荧光聚合酶链式反应技术检测食源性沙门氏菌和志贺氏菌

Detection of Salmonella and Shigella using duplex fluorescence real-time polymerase chain reaction with TaqMan probe

中文关键词:  沙门氏菌  志贺氏菌  食源性  荧光PCR

英文关键词:Salmonella  Shigella  food-borne  fluorescent polymerase chain reaction

中文摘要:

      目的 建立TaqMan探针双重荧光聚合酶链式反应(polymerase chain reaction, PCR)技术检测食源性沙门氏菌和志贺氏菌的方法。方法 通过比对沙门氏菌和志贺氏菌的基因组序列, 选择同源性高、保守性好的区域设计特异性引物和探针, 经过引探筛选、浓度调试等一系列优化, 建立了食源性沙门氏菌和志贺氏菌双重荧光PCR核酸检测方法, 并对其特异性、质粒最低检出限、菌液敏感性、重复性、稳定性以及实际样品进行了验证。结果 该方法与大部分食源性致病菌无交叉反应, 特异性强; 沙门氏菌和志贺氏菌质粒的最低检出限可达5 copies/μL, 沙门氏菌菌液敏感性为1.0×102 cfu/mL, 志贺氏菌菌液敏感性10 cfu/mL; 质粒和菌液核酸梯度的批内和批间变异系数均在0.177%~1.958%之间, 小于5.0%, 具有较强的稳定性; 标准曲线相关系数(r2)均大于0.999。结论 本研究成功建立了一种TaqMan探针双重荧光PCR技术同时检测食源性沙门氏菌和志贺氏菌的方法, 该方法扩增时间短, 只需要30 min即可, 而且特异性强、稳定性好, 可用于疑似沙门氏菌和志贺氏菌污染样品的检测, 为食品安全的快速检测提供技术支撑。

英文摘要:

      Objective To establish a TaqMan probe dual fluorescence polymerase chain reaction (PCR) method for the detection of foodborne Salmonella and Shigella. Methods By comparing the genomic sequences of Salmonella and Shigella, selecting regions with high homology and good conserved design specific primers and probes, and through a series of optimization such as induction screening and concentration adjustment, a dual fluorescent PCR nucleic acid detection method for foodborne Salmonella and Shigella was established. The specificity, minimum limit of detection of plasmid, sensitivity, repeatability, stability and actual samples were verified. Results The method had no cross-reaction with most foodborne pathogens and had high specificity. The minimum limit of detection of Salmonella and Shigella plasmids were 5 copies/μL. The sensitivity of Salmonella fluid was 1.0×102 cfu/mL, and that of Shigella was 10 cfu/mL. The coefficient of variation of nucleic gradient in plasmid and liquid was 0.177% to 1.958%, less than 5.0%, which showed strong stability. The correlation coefficients (r2) of standard curves were all greater than 0.999. Conclusion In this study, a TaqMan probe dual fluorescent PCR method for simultaneous detection of food-borne Salmonella and Shigella is successfully established. The amplification time of the method is short, requiring only 30 min, and the method has strong specificity and good stability. It can be used for the detection of suspected Salmonella and Shigella contaminated samples, providing technical support for rapid detection of food safety.