荧光重组酶介导等温扩增快速检测食品中克罗诺杆菌属

Rapid detection of Cronobacter spp.in food by fluorescent recombinase-aided amplification

目的建立快速简便的荧光重组酶介导等温扩增法(recombinase-aided amplification,RAA)检测克罗诺杆菌属,以满足口岸快速通关及监管需要。方法根据克罗诺杆菌属ompA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,同时与GB 4789.40—2016《食品安全国家标准食品微生物学检验克罗诺杆菌属(阪崎肠杆菌)检验》进行比对验证。结果克罗诺杆菌属荧光RAA最佳反应温度为39℃,最佳引物、探针终浓度均为400 nmol/L。建立的荧光RAA法特异性强,纯菌灵敏度达到10^(2) CFU/mL。加标食品基质婴儿奶粉及婴儿米粉在改良月桂基硫酸盐胰蛋白胨肉汤-万古霉素(modified lauryl sulfate tryptose broth-vancomycin medium,mLST-Vm)增菌只需2 h,即可检测原始浓度达到10-2CFU/mL的克罗诺杆菌属。荧光RAA法只需20~30 min完成扩增,5 min即可观察结果,速度及灵敏度明显高于国家标准方法。结论荧光RAA法简便、快速、无需大型仪器,可用于口岸或其他场所进行克罗诺杆菌属的快速检测与监控。

Objective To establish a rapid and simple fluorescent recombinase-aided amplification(RAA)method to detect Cronobacteria spp.in order to meet the actual needs of port rapid customs clearance and supervision.Methods According to the conserved region of ompA gene of Cronobacter spp.,the specific primers and probes were designed,the primer combinations with the best amplification efficiency and sensitivity were selected by combining the primers and probes,the optimal reaction conditions were determined by optimizing the reaction temperature and the concentration of primers and probes.The established fluorescence RAA method was applied to the detection of food matrix and actual samples,and compared with the national standard GB 4789.40—2016 National Food Safety Standard-Food Microbiology Test-Cronobacter(Enterobacter sakazakii) test for verification.Results The optimum reaction temperature of fluorescent RAA for Cronobacter spp.was 39 ℃,and the final concentration of primer and probe was 400 nmol/L.The established fluorescence RAA method showed high specificity and the sensitivity,and the detection limit of the method was 10^(2) CFU/mL in pure culture.The limit of detection for Cronobacter spp.was 10-2 CFU/mL original concentrations under 2 h modified lauryl sulfate tryptose broth-vancomycin medium(mLST-Vm) enrichment in artificially contaminated baby milk powder and baby rice.The fluorescence RAA assay gave a positive signal in as early as 5 min,and the whole assay could be completed in approximately 20-30 min.The speed and sensitivity were significantly higher than those of the national standard method.Conclusion Fluorescence RAA method is simple,rapid and does not need large instruments,which can be used for rapid detection and monitoring of Cronobacteria spp.at ports or other places.