基于竞争性互补介导核酸恒温扩增技术快速检测沙门氏菌  

A Rapid Competitive Annealing Mediated Isothermal Amplification for Salmonella Detection

作　　者：陈旭[1] 毛瑞 李堂正 乔宇[4] 彭晴[4] 杜昱光[2] CHEN Xu;MAO Rui;LI Tang-zheng;QIAO Yu;PENG Qing;DU Yu-guang(College of Food Science,Shenyang Agricultural University,Shenyang 110161,China;State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 1001903,China;College of Life Science,Heilongjiang University,Harbin 150080,China;Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]沈阳农业大学食品学院,沈阳110161 [2]中国科学院过程工程研究所生化工程国家重点实验室,北京100190 [3]黑龙江大学生命科学学院,哈尔滨150080 [4]中国农业科学院饲料研究所,北京100081

出　　处：《沈阳农业大学学报》2019年第2期174-179,共6页Journal of Shenyang Agricultural University

基　　金：国家重点研发计划项目(2018YFD0501403)

摘　　要：食源性致病菌污染是食品安全问题的重要隐患之一,沙门氏菌是食品中最常见的食源性致病菌。为快速检测食品中的沙门氏菌,满足食品生产过程中实时检测监控的需要,基于竞争性互补介导核酸恒温扩增(competitive annealing mediated isothermal amplification,CAMP)技术,建立一种操作简便、准确性高、灵敏度高的沙门氏菌快速检测方法。以沙门氏菌invA基因保守区域片段为靶序列,利用DNAMAN设计特异性引物,并在己筛选获得沙门氏菌的CAMP引物基础上,应用CAMP引物设计推荐规则及作用位点,进行加速引物的设计和筛选,评估加速引物对扩增反应的影响;并且对建立的方法进行特异性及灵敏性的检测评估。同时与PCR方法进行比较。结果表明:以沙门氏菌invA基因为目标核酸所设计的一对CAMP引物可以实现对沙门氏菌的快速检测,而且在反应体系中引入加速引物可以将检测时间缩短15min;PCR方法的检测限为103CFU·mL-1,而本研究所建立的方法在65℃恒温条件下80min内检出沙门氏菌,检测限为10CFU·mL-1,检测效率和灵敏度均优于传统PCR方法,而且操作简便,无需精密、复杂的变温仪器;对3株沙门氏菌和10株非沙门氏菌进行特异性检测,3株阳性对照株检测全部为阳性,10株阴性对照菌检测全部为阴性,说明本试验所建立沙门氏菌快速检测方法具有良好的特异性,而且对沙门氏菌无漏检,具有较好的通用性。Food-borne pathogenic bacteria contamination is an important hidden dangers in food safety.Salmonella is the most common food-borne pathogenic bacteria in food.In order to rapidly detect Salmonella in food and meet the need of food production in the process of real-time monitoring,a rapid detection method for Salmonella was established based on competitive complementary mediated isothermal amplification (CAMP) technology to,which is simple,accurate and sensitive.A pair of gene-specific primers for the conservative invA gene were designed by DNAMAN.And based on the primer design rules of CAMP,the accelerated primers were designed,and the effect of accelerated primers in isothermal amplification reaction was evaluated.The specificity and sensitivity assessment of this method was carried out.At the same time,it was compared with PCR.Theresults shown that the CAMP primers designed in this study base on the invA gene of Salmonella could be used for rapid detection of Salmonella,and accelerated primers could shorten the detection time by 15 min.The minimum detection limit of Salmonella was 103 CFU·mL^-1 by PCR.The method established in this study could be detected within 80 min at 65 ℃.And the detection limit of the method was 10 CFU·mL^-1,which is better than traditional PCR method.Moreover,the method is simple to operate and does not require precise and complicated temperature-changing instruments.The specific detection of three Salmonella strains and ten non-Salmonella strains showed that all the three Salmonella strains were detected without non-specific amplification of the ten non-Salmonella strains.It showed that the rapid detection method of Salmonella established in this study had good specificity and versatility.

关 键 词：沙门氏菌 竞争性互补介导核酸恒温扩增 快速检测 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]