4种常见食源性病原菌多重PCR检测技术研究  

Study on multiple PCR detection techniques for four common foodborne pathogens

作　　者：王珊[1] 王雁伟 高辉明 艾鹏飞[1] WANG Shan;WANG Yanwei;GAO Huiming;AI Pengfei(College of Food Science and Biology,Hebei University of Science and Technology,Shijiazhuang,Hebei 050018,China)

机构地区：[1]河北科技大学食品与生物学院,河北石家庄050018

出　　处：《河北工业科技》2024年第2期133-140,共8页Hebei Journal of Industrial Science and Technology

基　　金：河北省科技计划“奶业振兴重大技术创新专项”(19227137D);河北省大中学生科技创新专项(22E50074D)。

摘　　要：为了解决常规方法检测食源性病原菌存在操作繁琐、周期长的问题,基于4种常见食源性病原菌特异基因序列扩增提出了一种快速、高效的多重PCR(multiplex polymerase chain reaction, MPCR)检测方法。根据单增李斯特菌iap基因、蜡样芽孢杆菌gyrB基因、产志贺毒素大肠埃希氏菌stxⅠ基因、肠炎沙门氏菌invA基因的特异序列分别设计引物后建立MPCR反应体系,测试其特异性、敏感性和可行性,并与国标培养法进行比较。结果表明:MPCR扩增出4个目标基因的特异性条带大小依次为371、221、432、171 bp;在58℃的退火温度下,MPCR扩增表现出良好的特异性,无非特异性扩增,4种病原菌最低检出限达到102CFU/mL;MPCR方法和国标培养法对多种食源性病原菌感染的牛奶样品的检测结果完全一致,检测周期由原来的5~7 d缩减到8~9 h。所建立的MPCR技术作为一种快速、高效的检测方法,可为食品安全生产提供保障。The method of efficient multiplex polymerase chain reaction(MPCR)was constructed based on the amplification of specific gene sequences of four common food borne pathogens in order to solve the problems of those traditional detection methods involving cumbersome operations and time consuming.The MPCR method was established using the designed primers according to the specific sequences of iap gene in Listeria monocytogenes,gyrB gene in Bacillus cereus,stxⅠgene in Shiga toxin-producing Escherichia coli,invA gene in Salmonella enteritidis,respectively.The specificity,the sensitivity and the feasibility of MPCR were analyzed,and it was compared with the national standard culture method.The results show that the specific bands of four target genes amplified by MPCR were 371,221,432 and 171 bp,respectively.Under the annealing temperature of 58℃,the MPCR presented strong specificity without non-specific amplification,and the lowest detection limit(LOD)of four pathogenic bacteria reached up to 102 CFU/mL.The two detecting results of the MPCR and the standard culture method were identical,and the detection period of MPCR was reduced to 8~9 h from that of traditional methods for 5~7 d.The constructed MPCR,as a rapid and efficient detecting method for food products in practice,provided guarantees for food safety.

关 键 词：食品检验学 多重PCR(MPCR) 单增李斯特菌 蜡样芽孢杆菌 产志贺毒素大肠埃希氏菌 肠炎沙门氏菌 

分 类 号：TS207.4[轻工技术与工程—食品科学]