环介导等温扩增方法在食品中沙门菌检测的应用和评价

Application and evaluation of loop-mediated isothermal amplification method for detceting of Salmonella spp. in food

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 The loop-mediated isothermal amplification （LAMP） detection method was applied to detect Salmonella spp. in food. The specificity and sensitivity of this method were compared with real-time PCR and conventional detection method. Methods ThefimY gene of Salmonella spp. was used to design LAMP primers, and then optimized LAMP reaction system. LAMP method was compared with real-time PCR and conventional detection methods in some aspects, such as specificity, sensitivity and practical food samples detection. Results The specificity of LAMP method was tested by using 93 targets and 31 non-targets bacteria. The results showed that the LAMP method was highlyspecific to Salmonella spp.. No cross-reaction was founded. In pure culture, the sensitivity of LAMP was 6.4 ×102cfu/ml, which was consistent with real-time PCR method. The detection limit of LAMP reached 2 cfu/25 g in base-material addition test. The detection of 45 practical food samples showed the detection rate of LAMP was 11.1% , which was as same as real-time PCR and traditional methods. Conclusion The LAMP detection method of Salmonella spp. established in this study has good specificity and sensitivity, which can apply to the rapid detection of Salmonella spp.