牡蛎中单核细胞增生李斯特菌PCR快速检测方法的建立  

Development of PCR rapid detection method for Listeria monocytogenes in oysters

作　　者：王晓芳 张若鸿 王纯 阎贺静[1] 姚红 刘绍军[1] 李晓然 杨洋 崔生辉[2] 郭云昌 Wang Xiaofang;Zhang Ruohong;Wang Chun;Yan Hejing;Yao Hong;Liu Shaojun;Li Xiaoran;Yang Yang;Cui Shenghui;Guo Yunchang(College of Food Science and Technology,Hebei Normal University of Science and Technology,Qinhuangdao 066600,China;China National Institutes for Food and Drug Control,Beijing 100050,China;China National Center for Food Safety Risk Assessment,Beijing 100022,China)

机构地区：[1]河北科技师范学院食品科技学院,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：《卫生研究》2023年第2期265-271,共7页Journal of Hygiene Research

基　　金：河北省自然科学基金面上项目(No.H2022407001,H2017407007);河北省现代农业产业技术体系露地蔬菜创新团队项目(No.HBCT2021200207);河北省重点研发计划(No.19222810D);河北省高等学校科学技术研究青年基金(No.QN2016256)。

摘　　要：目的建立一种无需前增菌快速检测牡蛎中单核细胞增生李斯特菌的PCR方法。方法利用β-环糊精和膨润土包被活性炭处理牡蛎样品,去除聚合酶链反应(polymerase chain reaction,PCR)反应抑制因子,以单核细胞增生李斯特菌特异基因inlB为靶基因,建立无需样品前增菌的PCR快速检测方法。同时验证该方法的特异性、灵敏度及实用性参数,并评估该方法所用试剂在冷冻保存后的稳定性与实用性。结果建立的PCR方法对130株目标菌和63株非目标菌的检测特异性为100%,灵敏度达10 CFU/25 g。该方法无需前增菌,4 h即可完成检测。PCR方法和国标方法对实际样品中单核细胞增生李斯特菌的检出率(均为13%)和活菌检测率(均为100%)一致,即符合率100%。此外,该方法所用试剂在-20℃冷冻条件下可稳定保存至少1年。结论建立的PCR方法特异性强、灵敏度高,可快速、准确检测出牡蛎中的单核细胞增生李斯特菌。OBJECTIVE To develop a polymerase chain reaction(PCR)method for rapid detection of Listeria monocytogenes in oysters without pre-enrichment.METHODS The combination ofβ-cyclodextrin and bentonite-coated activated carbon was used to remove PCR inhibitors from oyster samples,and the target gene inlB was used for the PCR subsequently.The specificity,sensitivity,and application of the developed method were verified,and the stability and application of the reagents stored under cryopreservation conditions were evaluated.RESULTS The specificity of the developed PCR method was 100%for the detection of 130 target bacterial strains and 63 non-target bacterial strains.The method reduced the time required for Listeria monocytogenes detection to 4 h without pre-enrichment,and the detection limit was 10 CFU/25 g.The method was consistent with the conventional culture method on the detection rate and viable bacteria detection rate of Listeria monocytogenes in natural oyster samples(the coincidence rate was 100%).Additionally,the reagents could be used normally after storing at-20℃for at least one year.CONCLUSION The PCR method developed in this study has high specificity and sensitivity,and can be used for rapid,accurate detection of Listeria monocytogenes in oysters.

关 键 词：单核细胞增生李斯特菌 Β-环糊精 膨润土包被活性炭 牡蛎 聚合酶链反应 

分 类 号：R155.5[医药卫生—营养与食品卫生学]