生鲜乳中16种致病微生物多重荧光定量PCR集成检测技术的建立与初步应用  

Establishment and Application of Multiplex Integrated qPCR System for 16 Kinds of Pathogenic Microorganisms in Raw Milk

作　　者：肖沙 赵格[2,3,4] 赵建梅 向祝[1,2,3] 宋时萍 刘娜 张喜悦 徐莹[1] 王君玮 Xiao Sha;Zhao Ge;Zhao Jianmei;Xiang Zhu;Song Shiping;Liu Na;Zhang Xiyue;Xu Ying;Wang Junwei(College of Food Science and Engineering,Ocean University of China,Qingdao 266000,Shandong,China;China Animal Health and Epidemiology Center,Qingdao 266032,Shandong,China;Laboratory of Quality and Safety Risk Assessment for Livestock and Poultry Products(Qingdao),Ministry of Agriculture and Rural Affairs,Qingdao 266032,Shandong,China;Key Laboratory of Animal Biosafety Risk Warning and Prevention and Control(South China),Ministry ofAgriculture and Rural Affairs,Qingdao 266032,Shandong,China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266000 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]农业农村部畜禽产品质量安全风险评估实验室(青岛),山东青岛266032 [4]农业农村部动物生物安全风险预警及防控重点实验室(南方),山东青岛266032

出　　处：《中国动物检疫》2024年第7期86-95,116,共11页China Animal Health Inspection

基　　金：国家重点研发计划项目(2022YFD1301003)。

摘　　要：为满足生鲜乳中微生物快速通量检测的需求,建立多重荧光定量PCR集成方法,从而快速检测生鲜乳中潜在的16种致病微生物,通过设计致病性大肠杆菌、金黄色葡萄球菌、链球菌等目标菌株的特异性引物和探针,优化反应体系,验证方法的特异性、敏感性等,建立了稳定的4组多重集成荧光定量PCR反应体系,并利用人工污染的生鲜乳样品对所建立的方法和传统培养法进行了验证比较。结果显示:各对引物探针对目标菌株均能有效识别并扩增,每组体系中引物探针未发现交叉反应,对其余3组体系的12株非目标菌均未有特异性反应;组内和组间变异系数均低于3%;该方法对布鲁氏菌的最低检出限为10^(2) CFU/mL,对阪崎肠杆菌、志贺菌、沙门菌等7种致病微生物的最低检出限为10^(3) CFU/mL,对蜡样芽胞杆菌、弯曲杆菌、结核分枝杆菌等8种致病微生物的最低检出限为10^(4) CFU/mL;所建方法和传统培养法对人工污染不同致病菌的各25份样品检测结果基本一致。结果表明,本研究建立的多重集成荧光定量PCR方法灵敏度高,特异性及重复性好,可实现对生鲜乳样品中多种致病微生物的快速高效检测,为生鲜乳微生物性风险监测提供了技术支撑。s:In order to meet the need of rapidly detecting microorganisms in raw milk at a high throughput,a multiplex integrated real-time quantitative PCR(qPCR)was established to rapidly detect 16 pathogenic microorganisms that might be available in raw milk,specific primers and probes for the target strains including pathogenic Escherichia coli,Staphylococcus aureus,Streptococcus spp.and other bacteria were designed,after optimizing the reaction system and evaluating the specificity and sensitivity of the method,a stable 4-group multiplex qPCR system was established,and compared with dot immunogold filtration assay(DIGFA)using artificially contaminated raw milk samples.The results showed that all the primers and probes could effectively amplify targeted strains,without crossreaction in each group or specific reaction to 12 non-targeted strains in other 3 groups the coefficients of variation(CVs)of the intra-and inter-groups were both lower than 3%:its lowest detection limit was 10^(2) CFU/mL for Brucella,10^(3) CFU/mL for 8 pathogenic microorganisms such as Enterobacter sakazaki,Bacillus cereus and Salmonella,and 10^(4) CFU/mL for 7 pathogenic microorganisms such as Shigella,Campylobacter and Streptococcus;for each of 25 samples artificially contaminated with pathogenic bacteria,the test results by the established method were basically consistent with those by DIGFA.In conclusion,the established multiplex integrated qPCR system,with good sensitivity,specificity and reproducibility,could be used for rapid and efficient detection of various pathogenic microorganisms in raw milk samples,supporting the microbiological risk monitoring of raw milk.

关 键 词：致病微生物 多重荧光定量PCR 生鲜乳 集成检测技术 

分 类 号：S852.43[农业科学—基础兽医学]