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目的将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×102cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2 cfu/25 g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。
OBJECTIVE: To apply loop-mediated isothermal amplification assay to the detection of Salmonella in food and compare it with real-time PCR and traditional detection methods in terms of specificity and sensitivity. Methods Based on the highly conserved fimY gene of Salmonella, loop-mediated isothermal amplification (PCR) primers were designed and the reaction system was optimized. Compared with real-time PCR and conventional detection methods, the primers were optimized for specificity, sensitivity and real sample detection. Results The LAMP method established in this study detected 93 Salmonella strains and 31 non-target bacteria strains with good specificity. In pure culture, without increasing bacteria, the detection sensitivity of 6.4 × 102cfu / ml, and real-time fluorescence PCR method. In the food matrix addition test, the detection limit of 2 cfu / 25 g was determined by ring-mediated isothermal amplification method. The detection results of 45 actual food samples showed that the actual detection rate of this method was 11.1% The traditional method has the same test result. Conclusion The Salmonella ring-mediated isothermal amplification assay established in this study has good specificity. The detection sensitivity is comparable to that of real-time PCR and is suitable for the rapid screening of Salmonella.