荧光重组酶介导等温扩增快速检测食品中沙门氏菌

Detection of Salmonella in Food by Fluorescent Recombinase Mediated Isothermal Amplification

针对口岸食品检验中沙门氏菌传统检测法时间长的缺点,建立快速简便的荧光重组酶介导等温扩增法(recombinase-aided amplification,RAA),以适应口岸快速通关的实际需求。根据沙门氏菌inv A基因保守区设计特异性引物、探针,通过引物两两组合结合探针筛选出扩增效率及灵敏度最佳的引物组合,优化反应温度及引物探针浓度,确定最佳反应条件。将建立的荧光RAA法应用于食品基质及实际样品检测中,同时与GB 4789.4—2016进行比对验证。结果表明,沙门氏菌荧光RAA最佳反应温度为39℃,最佳引物、探针终浓度分别为200 nmol/L和400 nmol/L。建立的荧光RAA法特异性强,纯菌灵敏度达到3 CFU/mL,基质增菌前灵敏度为3×10^(3) CFU/mL。TTB增菌只需2 h,即可达到3×10^(-2) CFU/mL,高于传统培养法的3 CFU/mL。实际样品检测结果与国标法吻合。荧光RAA法只需5 min即可观察结果,20~30 min扩增效果最佳。相比于其他分子生物学方法,简便、快速、无需大型仪器,充分满足口岸或其他场所进行食源性微生物快速检测与监控。

In order to overcome the shortcomings of the traditional detection method of Salmonella in port food inspection for a long time,a fast and simple method of recombinase-aid amplification(RAA)was established to meet the actual needs of rapid customs clearance at ports.According to the conserved region of Salmonella inv A gene,the specific primers and probes were designed.Through the combination of primers and probes,the primer combination with the best amplification efficiency and sensitivity was selected.The reaction temperature and primer probe concentration were optimized to determine the best reaction conditions.The established fluorescence RAA method was applied to the detection of food matrix and actual samples,and compared with the national standard GB 4789.4—2016.The results revealed that the optimal reaction temperature was 39℃,and the optimal final concentration of primer and probe were 200 nmol/L and 400 nmol/L,respectively.The fluorescence RAA method was specific,the sensitivity of pure bacteria was 3 CFU/mL,and that the sensitivity of substrate before enrichment was 3×10^(3) CFU/mL.The sensitivity of TTB enrichment can reach 3×10^(-2) CFU/mL in 2 h,higher than 3 CFU/m L of traditional culture method.The results of the actual samples were consistent with the national standard method.Fluorescence RAA method only needs 5 minutes to observe the results,and 20-30 minutes was the best.Compared with other molecular biological methods,the method was simple,rapid and did not need large-scale instruments,which could fully meet the requirements of rapid detection and monitoring of foodborne microorganisms at ports or other places.