四重数字PCR检测乳及乳制品中致病菌  

Quadruplex digital PCR for detection of pathogenic bacteria in milk and dairy products

作　　者：王越 张奕南[1] 刘洋[1] WANG Yue;ZHANG Yinan;LIU Yang(Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology,State Administration for Market Regulation,shanghai Institute of Quality Inspection and Technical Research,Shanghai 200233,China)

机构地区：[1]国家市场监督管理总局重点实验室(乳及乳制品检测与监控技术)上海市质量监督检验技术研究院,上海200233

出　　处：《中国乳品工业》2025年第3期61-69,共9页China Dairy Industry

基　　金：上海市科委项目(21N31900700)。

摘　　要：为了满足乳制品中食源性致病菌精确、高效和高通量检测需求,文章以典型食源性致病菌大肠埃希氏菌O157、金黄色葡萄球菌、单核细胞增生李斯特氏菌和沙门氏菌作为目的菌,建立1种灵敏稳定的多重芯片式数字PCR(Multiplex digital chip PCR)反应体系。针对4种致病菌的靶向基因,设计特异性引物,优化多重反应引物组合和反应条件,并验证其特异性和灵敏度。结果表明,该四重反应体系具有良好的特异性且未出现交叉反应。体系对4种菌株最低检出限分别达0.38、1.03、0.69 copies/μL和0.24 copies/μL,与单重反应体系检测灵敏度相当,未发生混合引物抑制反应。方法对模拟阳性样品、生乳和市售乳制品检测结果证明了其在乳原料及乳制品质量安全控制中的应用前景,文章构建的四重反应体系可为乳制品的检测与监管提供技术支持。In order to meet the precise and efficient,and high-throughput detection needs of foodborne pathogenic bacteria in dairy products,this study aimed to establish a sensitive and stable multiplex digital chip Polymerase Chain Reaction system targeting the typical foodborne pathogenic bacteria Escherichia coli O157:H7,Staphylococcus aureus,Listeria monocytogenes,and Salmonella,as typical foodborne pathogenic bacteria.Specific primers were designed for the target genes of the four pathogens,and the primer combinations and reaction conditions for the multiplex reaction were optimized and the specificity and sensitivity of reaction were verified.The results demonstrated that the quadruplex reaction system demonstrated good specificity with no cross-reactivity.The system achieved the lowest detection limits for the four bacterial strains at 0.38,1.03,0.69 copies/μL,and 0.24 copies/μL,comparable to the sensitivity of singleplex reaction systems,without encountering multiplex primer inhibition.The result of simulated positive samples,raw milk and commercial dairy products validated the potential application of this method in ensuring the quality and safety of dairy products.The quadruplex reaction system developed in this study can provide technical support for the detection and regulation of dairy products.

关 键 词：多重数字PCR 食源性致病菌 检验检测 乳制品 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]