等温扩增技术在食品中沙门氏菌检测中的应用

Application of isothermal amplification technique in the detection of Salmonella in food

目的针对沙门氏菌肠侵袭蛋白基因设计适合交叉引物等温扩增法及环介导等温扩增法的特异性引物及探针,并进行应用评估。方法用19株沙门氏菌,35株近源菌进行特异性试验;通过定量DNA、纯菌液计数检测进行检测低限验证,与环介导等温扩增法进行比较;对6类18种食品根据ISO 5725-2:1994标准要求,以现行国家标准为基准方法对2种等温扩增法进行灵敏度、特异性、假阳性率、假阴性率及相对准确度等5方面验证。结果交叉引物等温扩增法与环介导等温扩增法具有相同的特异性,检测灵敏度较高于环介导等温扩增法,与基准方法比较的各项性能指标符合ISO的要求。结论该方法不需要复杂仪器,可作为食品中沙门氏菌快速定性检测方法使用,对口岸快速筛查、基层或偏远经济不发达地区顺利开展检测具有实际意义。

Objective To design the specific primers and probes on the basis of sequence analysis of Salmonella, and evaluate the application of isothermal amplification methods（cross-priming amplification and loop-mediated isothermal amplification, CPA and LAMP） in the detection of Salmonella in food. Method The specificity of the method was evaluated by 19 strains of Salmonella and 35 negative strains of Enterobacteriaceae. The sensitivity of the CPA method was evaluated by testing the quantitative DNA and pure bacteria count compared with the LAMP method. According to the ISO standard, 2 isothermal amplification methods were confirmed from the aspects of sensitivity, specificity, false positive rate, false negative rate and relative accuracy with the current national standard as the reference method. Result The CPA method had the same specificity as the LAMP method, was more sensitive than the LAMP method. The performance of methods could meet the requirements of ISO which compared with the reference method. Conclusion This method is highly sensitive, specific, and does not require any complex equipment, which can be used as a rapid qualitative detection method for Salmonella in food. It has practical significance for the rapid screening in the ports, and the detection in grassroots areas or the economically underdeveloped areas in remote areas.