实时荧光定量PCR法鉴定发酵乳中嗜热链球菌  

Identification of Streptococcus thermophilus in fermented milk by real-time fluorescent quantitative PCR

作　　者：王青龙 貌达 周燕霞 刘凯 李爽 杨霞 王雨婷 王军红 蔡雪凤 WANG Qinglong;MAO Da;ZHOU Yanxia;LIU Kai;LI Shuang;YANG Xia;WANG Yuting;WANG Junhong;CAI Xuefeng(The Center for Supervision and Inspection of Food Quality and Safety of Beijing(Food Inspection Institute of Beijing),Beijing 100094,China;Division of Chemical Metrology and Analytical Science,National Institute of Metrology,Beijing 100029,China)

机构地区：[1]北京食品安全监控和风险评估中心(北京市食品检验所),北京100094 [2]中国计量科学研究院化学计量与分析科学研究所,北京100029

出　　处：《中国酿造》2022年第7期149-154,共6页China Brewing

基　　金：国家市场监督管理总局科技计划项目(2019MK004)。

摘　　要：该研究通过系统发育树分析确定目标基因,根据目的基因设计特异性引物和探针,建立一种能够快速准确鉴定发酵乳中嗜热链球菌的实时荧光定量聚合酶链式反应(RT-fqPCR)法,通过特异性、灵敏性和抗干扰实验对所建立方法进行验证,并使用该方法对市售的60份标识含有嗜热链球菌的发酵乳样品进行检测。结果表明,recA基因具有种间特异性,种间差异率>10%,以其为目的基因建立的RT-fqPCR方法能够特异性的检测嗜热链球菌;绝对灵敏度达1 pg/μL,相对灵敏度达10^(3) CFU/mL;在培养物水平和基因组水平抗干扰能力良好。采用该方法从60份标识含有嗜热链球菌的发酵乳样品中均能检测出嗜热链球菌,说明实时荧光定量PCR方法能够快速、准确的对发酵乳中嗜热链球菌进行检测。Target genes were identified by phylogenetic tree analysis, and specific primers and probes were designed according to the target gene to establish a real-time fluorescence quantitative polymerase chain reaction (RT-fqPCR) method that could quickly and accurately identify Streptococcus thermophilus in fermented milk. The proposed method was verified by specificity, sensitivity and anti-interference tests, and 60 commercial fermented milk products labeled with S. thermophilus were tested by the method. The results showed that recA gene had specificity between species, and the difference rate between species was greater than 10%. The RT-fqPCR method established with recA gene as the target gene could specifically detect S. thermophilus, the absolute sensitivity was 1 pg/μl, the relative sensitivity was 10^(3) CFU/ml, and the anti-interference ability at culture level and genome level was good. S. thermophilus could be detected in all 60 samples of fermented milk labeled with S. thermophilus using this method, indicating that real-time fluorescence quantitative PCR method could detect S. thermophilus in fermented milk quickly and accurately.

关 键 词：实时荧光定量PCR 嗜热链球菌 RECA基因 发酵乳 分子鉴定技术 

分 类 号：Q93-331[生物学—微生物学]