4种致泻大肠埃希氏菌数字PCR检测方法的建立  

Establishment of Digital PCR Assay to Detect Four Strains of Diarrheagenic Escherichia Coli

作　　者：马丽侠[1] 周李华[1] 叶德萍[1] 蒋子敬 姜展樾 易艳 MA Lixia;ZHOU Lihua;YE Deping;JIANG Zijing;JIANG Zhanyue;YI Yan(Institute of Biology,National Institute of Measurement and Testing Technology,Chengdu 610021,China;College of Food,Sichuan Agriculture University,Ya’an 625000,China)

机构地区：[1]中国测试技术研究院,生物研究所,四川成都610021 [2]四川农业大学食品学院,四川雅安625000

出　　处：《食品科技》2022年第9期290-296,共7页Food Science and Technology

基　　金：国家重点研发计划项目(2021YFF0701905)。

摘　　要：针对常见致泻大肠埃希氏菌特征基因stx2、escV、lt、pic,研究建立了一套微滴数字PCR(ddPCR)方法。优化了引物探针浓度,退火温度等条件,考察了方法特异性、线性范围、精密度。结果表明,特征基因stx2在2.0~24506.67个拷贝,escV在8.8~24756.67个拷贝,lt在1.63~19990.00个拷贝,pic在1.63~26500.00个拷贝均呈线性关系,线性系数均大于0.99;确定了各基因的定量限(stx2、escV、lt、pic分别为21个拷贝、20.60个拷贝、21.67个拷贝、27.67个拷贝);方法精密度小于10%,特异性良好。采用该方法对实际样品进行检测,结果显示,加标样品显示有阳性微滴,而其余样品均未检出阳性微滴,表明建立的数字PCR方法可用于食品中致泻大肠埃希氏菌的检测。该方法可为监管部门对生产牛肉制品、即食生肉制品、生食果蔬制品等重点企业生产环境卫生的检查提供技术支撑,有利于提高我国食品生产中原料、环境及加工过程的卫生水平,防止微生物污染和交叉污染,为食品生产全链条进行食品安全风险防范提供有力保障。In this study,a droplet digital PCR(ddPCR)method was developed for the genes stx2,escV,lt and pic of diarrheagenic Escherichia coli.The concentration of primer probe and annealing temperature were optimized,and the specificity,linear range and precision of the method were investigated.The results show that the concentration ranges of the characteristic genes stx2 from 2.0 copies to 24506.67 copies;escV from 8.8 copies to 24756.67 copies;lt from 1.63 copies to 19990.00 copies;and pic from 1.63 copies to 26500.00 copies were linear,with linear coefficients greater than 0.99.The quantitative limit of stx2 was 21 copies,escV was 20.60 copies,lt was 21.67 copies and pic was 27.67 copies.The precision was better than 10%and the specificity was good.The results showed that the samples were tested by the ddPCR,and the artificially contaminated samples showed positive,while the rest of the samples showed negative,indicating that the digital PCR method can be used for the detection of diarrhea-causing Escherichia coli in food.It can provide technical support for the regulatory authorities to inspect the environmental of enterprises such as beef products,instant raw meat and raw fruit or vegetable products,improve the hygiene level of raw materials,environment and processing of food production and operation in China,prevent microbial pollution and cross contamination,and provide a strong guarantee for food safety risk prevention throughout the food production chain.

关 键 词：大肠埃希氏菌 致泻大肠埃希氏菌 质粒DNA 微滴数字PCR 

分 类 号：TS207.4[轻工技术与工程—食品科学]