叶类蔬菜中单核细胞增生李斯特氏菌PCR快速检测方法的建立  

Rapid Detection of Listeria monocytogenes in Leafy Greens Based on PCR

作　　者：李晓然 张若鸿 王纯 尹树仁 王晓芳 杨洋 崔生辉[2] 郭云昌 LI Xiaoran;ZHANG Ruohong;WANG Chun;YIN Shuren;WANG Xiaofang;YANG Yang;CUI Shenghui;GUO Yunchang(College of Food Science and Technology,Hebei Normal University of Science and Technology,Qinhuangdao,Hebei 066600;National Institutes for Food and Drug Control,Beijing 100050;China National Center for Food Safety Risk Assessment,Beijing 100022)

机构地区：[1]河北科技师范学院食品科技学院,河北秦皇岛066600 [2]中国食品药品检定研究院,北京100050 [3]国家食品安全风险评估中心,北京100022

出　　处：《核农学报》2022年第10期2009-2018,共10页Journal of Nuclear Agricultural Sciences

基　　金：河北省自然科学基金面上项目(H2017407007);河北省高等学校科学技术研究青年基金项目(QN2016256);秦皇岛市科技支撑项目(201701B041);河北科技师范学院博士研究启动基金项目(2015YB010)。

摘　　要：为建立一种无需样品前增菌即可快速检测叶类蔬菜中单核细胞增生李斯特氏菌的聚合酶链式反应(PCR)方法,本试验将β-环糊精和牛乳蛋白包被活性炭结合用于去除叶类蔬菜基质中的PCR反应抑制因子,从而促进该菌的回收,随后以iap为靶基因,进行PCR检测,确定该方法的特异性、灵敏度和实用性,并评估该方法所用主要试剂在冷冻条件下的稳定性。结果表明,与prfA序列相比,该方法检测78株目标菌和63株非目标菌后,无假阳性或假阴性结果,特异性为100%;该方法无需前增菌,可在4 h内完成检测,灵敏度为10~1 CFU·25g^(-1);该方法与常规培养法在实际叶类蔬菜样品中目标菌的检出率、活体菌检测率均一致(符合率100%);所用的主要试剂在-20℃冰箱中保存至少12个月后仍可正常使用,稳定性较好。综上,该方法可快速、灵敏、特异地检测叶类蔬菜中的单核细胞增生李斯特氏菌,且实用性较好。本研究结果为降低单核细胞增生李斯特氏菌对消费者造成的安全风险提供了技术支持。To develop a PCR-based method for rapid detection of Listeria monocytogenes(L.monocytogenes)in leafy greens without sample pre-enrichment,the combination ofβ-cyclodextrin and milk protein-coated activated charcoal was used to remove the PCR inhibitors from leafy green matrices and facilitate the recovery of the target pathogen,and the target gene(iap)was used for PCR subsequently.The specificity,sensitivity,and application of the method were determined,and the stability of the major reagents used in the method under freezing conditions were evaluated.The results showed that there were no false positive or negative results for 78 target bacterial strains and 63 non-target bacterial strains after using the method(the specificity of the method was 100%)compared with the prfA sequence;the method achieved the detection within 4 h without pre-enrichment,and the sensitivity of the method was 10~1 CFU·25 g;the detection rate of L.monocytogenes and the detection rate of viable bacteria in natural leafy green samples were consistent in the method and the conventional culture method(the coincidence rate was 100%);the major reagents used in the method could be used normally after storing at-20℃for at least 12 months,which proved that these reagents have good stability.This method for the detection of L.monocytogenes in leafy greens was rapid,sensitive,and specific,and the application of the method was good,which can provide technical support for reducing the safety risk of consumers caused by this pathogen.

关 键 词：单核细胞增生李斯特氏菌 Β-环糊精 牛乳蛋白包被活性炭 叶类蔬菜 PCR 

分 类 号：TS255.7[轻工技术与工程—农产品加工及贮藏工程]