婴儿配方乳粉中食源性致病菌双重ERA快速检测方法的建立  

Establishment of a Dual Enzymatic Recombinase Amplification Method for Rapid Detection of Foodborne Pathogens in Infant Formula Powder

作　　者：林志伟[1] 王帅 王迎春 吴占文 李涛[2] 李红娜[2] 杨艳歌[2] 袁飞[2] LIN Zhiwei;WANG Shuai;WANG Yingchun;WU Zhanwen;LI Tao;LI Hongna;YANG Yange;YUAN Fei(College of Agriculture,Heilongjiang Bayi Agricultural University,Daqing 163000,China;Key Laboratory of Food Quality and Safety for State Market Regulation,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163000,China)

机构地区：[1]黑龙江八一农垦大学农学院,黑龙江大庆163000 [2]中国检验检疫科学研究院,国家市场监管重点实验室(食品质量与安全),北京100176 [3]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163000

出　　处：《食品科学》2023年第18期347-354,共8页Food Science

基　　金：“十三五”国家重点研发计划重点专项(2022YFF0607900;2018YFC1603606)。

摘　　要：为快速多重筛查婴儿配方乳粉中食源性致病菌的污染情况,建立克罗诺杆菌、沙门氏菌、单核细胞增生李斯特菌及金黄色葡萄球菌双重酶促重组等温扩增(enzymatic recombinase amplification,ERA)快速检测方法。首先,通过筛选确定对4种食源性致病菌特异性好、扩增效率高的引物探针。经过优化建立两组双重ERA检测体系可快速筛查4种食源性致病菌。该方法对沙门氏菌、单核细胞增生李斯特菌及金黄色葡萄球菌的检出限均为10^(-2) ng/μL;对克罗诺杆菌的检出限为1 ng/μL。模拟污染实验显示,污染量为1 CFU/mL的样品增菌培养6 h后,可检出4种致病菌。应用该方法对市售婴儿配方乳粉样品进行检测,检测结果与行业标准规定的实时荧光聚合酶链式反应(polymerase chain reaction,PCR)法一致,证实了本研究建立的双重ERA检测方法的准确性和可靠性。采用本方法仅需20 min左右即可一次性检出4种致病菌,相较于传统的“金标准”方法以及实时荧光PCR法显著提高了检测效率,对于推进食源性致病菌的快速筛查具有重要意义。For quick and multiple detection of the contamination of foodborne pathogens in infant milk powder,a dual enzymatic recombinase amplification(ERA)method for the rapid detection of Cronobacter,Salmonella,Listeria monocytogenes and Staphylococcus aureus were established in this study.Firstly,specific and efficient primers and probes were selected for these foodborne pathogens.Then,two groups of dual-ERA systems were established and optimized.The minimum detection limit was 10^(-2) ng/μL for Salmonella,L.monocytogenes and S.aureus,and 1 ng/μL for Cronobacter.The results of simulated contamination test showed that the four foodborne pathogens could be detected simultaneously after 6 hours of enrichment culture at an initial contamination level of 1 CFU/mL.When this method and real-time polymerase chain reaction(PCR)were used to detect commercial infant milk powder,consistent results were obtained,which confirmed the accuracy and reliability of the dual-ERA method.This method could simultaneously detect the four foodborne pathogens in about 20 minutes.Compared with the traditional method and real-time PCR,the detection efficiency was significantly improved,which is of great significance for promoting the rapid screening of foodborne pathogens.

关 键 词：酶促等温扩增技术 克罗诺杆菌 沙门氏菌 单核细胞增生李斯特菌 金黄色葡萄球菌 

分 类 号：TS207.4[轻工技术与工程—食品科学]