重大活动食品安全保障中沙门氏菌现场检测方法的建立

Establishment of on-site testing methods for Salmonella in food safety assurance of major events

目的针对重大活动食品安全保障工作对于现场检测的时间短和设备简单的要求及供应食品的类型,建立经加热处理和未经加热处理食品的沙门氏菌检测方法。方法将重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)与侧流免疫层析法(lateral flow immunoassay,LFS)结合,建立RPA-LFS方法对切片水果样品进行检测,再加上叠氮溴化丙锭(propidium monoazide,PMA)处理,建立PMA-RPA-LFS方法,对熟肉制品进行检测。并通过检测人工污染样品及实际样品验证方法的适用性。结果RPA-LFS的沙门氏菌纯菌检出限为2.0×10^(1)CFU/mL,新鲜水果基质检出限为2.0×10^(1)CFU/g。对人工污染样品和实际样品的检测结果与GB4789.4—2016《食品安全国家标准食品微生物学检验沙门氏菌检验》方法的结果一致。PMA-RPA-LFS的PMA处理方法为0.1%脱氧胆酸钠37℃处理20 min,加入10μg/mL PMA,室温避光孵育10 min,曝光15 min。PMA-RPA-LFS方法的纯菌检出限为2.0×102CFU/mL,同时可抑制浓度为105CFU/mL的死菌的扩增。对人工污染样品和实际样品的检测结果与GB4789.4—2016方法的结果一致。结论本研究建立的PMA-RPA-LFS能区分沙门氏菌死菌和活菌,适用于经加热处理的食品的检测。RPA-LFS与PMA-RPA-LFS结合使用,能更好地满足不同类型食品类别的检测需求,为重大活动食品安全保障工作中致病菌检测的研究积累数据。

Objective To establish detection methods for Salmonella in heated and unheated food according to the requirements of the on-site testing in major events,which including short time and simple equipment as well as suitable for different types of food supplied Salmonella.Methods By combining recombinase polymerase amplification(RPA)with lateral flow immunoassay(LFS),RPA-LFS was established for the detection of sliced fruit samples.In addition,propidium monoazide(PMA)treatment was used to establish PMA-RPA-LFS for the detection of cooked meat products.The applicability of the methods was verifided by detecting artificially contaminated samples and actual samples.Results The limit of detection in RPA-LFS was 2.0×10^(1) CFU/mL for pure bacteria and 2.0×10^(1) CFU/g for fresh fruit matrix.The detection results of artificially contaminated samples and actual samples were consistent with the results of GB 4789.4—2016 National food safety standards-Food microbiological examination:Salmonella.The procedure of PMA treatment method for PMA-RPA-LFS involved treating with 0.1%sodium deoxycholate at 37℃for 20 minutes at first,and then adding PMA with a concentration of 10μg/mL,followed by incubated at room temperature in dark for 10 minutes,and exposed for 15 minutes finally.The limit of detection for pure bacteria in the method was 2.0×102 CFU/mL,while inhibiting the amplification of dead bacteria at a concentration of 105 CFU/mL.The detection results of artificially contaminated samples and actual samples were consistent with the results of GB 4789.4—2016.Conclusion The PMA-RPA-LFS established in this study can distinguish between dead and live Salmonella bacteria,which is suitable for the detection of food after heating treatment.The combination of RPA-LFS and PMA-RPA-LFS can meet the detection needs of different types of food categories better,which accumulate data for research on pathogenic bacteria detection in food safety assurance of major events.