食品中沙门氏菌恒温隔绝式PCR检测方法的建立  

Establishment of Insulated Isothermal PCR Detection Method for Salmonella in Food

作　　者：杨若璇 佟尧 赵燕英[2,3] 汤承[2,3] 刘骥[1,2] 朱成林 曾英杰 于基成 唐俊妮[1,2] YANG Ruo-xuan;TONG Yao;ZHAO Yan-ying;TANG Cheng;LIU Ji;ZHU Cheng-lin;ZENG Ying-jie;YU Ji-cheng;TANG Jun-ni(College of Food Sciences and Technology,Southwest Minzu University,Chengdu 610041,China;Key Laboratory of Qinghai-Tibetan Plateau,Animal Genetic Resource Reservation and Utilization of Ministry of Education,Southwest Minzu University,Chengdu 610041,China;College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Key Laboratory of Biotechnology and Bioresources Utilization,Dalian Minzu University,Dalian 116600,China)

机构地区：[1]西南民族大学食品科学与技术学院,四川成都610041 [2]西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都610041 [3]西南民族大学畜牧兽医学院,四川成都610041 [4]大连民族大学生物技术与资源利用教育部重点实验室,沈阳大连116600

出　　处：《现代食品科技》2021年第9期285-293,共9页Modern Food Science and Technology

基　　金：国家重点研发计划项目(2018YFD0500500);四川省科技计划项目(2019YJ0261,2019JDJQ0017);生物技术与资源利用教育部重点实验室开放课题(KF2020008);西南民族大学中央高校基本科研业务费专项资金资助(2020NTD04)。

摘　　要：为弥补传统培养方法耗时长和现场检测步骤繁琐等缺陷,该研究建立了一种针对食品中沙门氏菌的恒温隔绝式PCR快速检测方法。根据沙门氏菌的invA基因设计特异性引物和探针,通过水浴法快速提取细菌DNA,优化引物、探针以及模板用量,建立了一种基于恒温隔绝式PCR快速检测沙门氏菌的方法,并对方法的特异性和灵敏度及稳定性进行评价,最后对比建立的方法与传统PCR方法、传统分离培养法对实际食品样品中沙门氏菌污染的检测效果。建立的恒温隔绝式PCR检测方法特异性好,灵敏度高且与其他细菌无交叉反应,最低检出限可达75 CFU/mL,可在6 h内完成检测实际食品样品中污染的沙门氏菌,传统PCR方法至少需12 h才能达到与之相同的检测效果,传统培养法验证了建立方法的准确性和可靠性。本研究建立的恒温隔绝式PCR方法更快速,且操作简便,适用于现场检测食品中污染的沙门氏菌。In order to make up for the shortcomings of traditional culture method such as long time-consuming and cumbersome on-site detection steps,an insulated isothermal PCR(iiPCR)rapid detection method for Salmonella in food was established.Specific primers and probes were designed according to the invA gene of Salmonella;bacterial DNA was quickly extracted by the water bath method;the amount of primers,probes and templates was also optimized.Then,a method for rapid detection of Salmonella based on iiPCR was established.The specificity,sensitivity and stability of the established method were evaluated.At the same time,the established method was compared with the traditional PCR method and the traditional culture method for detecting Salmonella contamination in actual food samples.The established iiPCR detection method had good specificity,high sensitivity,and no cross-reaction with other bacteria.The lowest detection limit could reach 75 CFU/mL.The established iiPCR method could detect Salmonella contaminated in actual food samples within 6 hours,while the same detection effect was achieved by traditional PCR methods at least 12 hours.The accuracy of iiPCR method was verified by the traditional culture method.The established iiPCR method for detecting Salmonella contaminated in food is suitable for field detection,more quickly and simpler.

关 键 词：沙门氏菌 invA基因 恒温隔绝式PCR 快速检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]