酸乳中葡糖醋杆菌赖解旋酶恒温基因扩增检测方法的建立-食品科学.PDF

※分析检测 Journal of Dairy Science and Technology 2017, Vol.40 No.2 30 酸乳中葡糖醋杆菌赖解旋酶恒温基因扩增 检测方法的建立 1 1,* 2 2 2 2 2 2, * 唐心怡 ，刘 波 ，张 涛 ，李永艳，张翠侠，王 赞，章晶晶，周 巍 （1.北京市第十二中学，北京 100071 ；2.河北省食品检验研究院，河北省食品安全重点实验室，河北 石家庄 050071 ） 摘  要：建立酸乳中葡糖醋杆菌的赖解旋酶恒温基因扩增（helicase-dependent isothermal DNA amplification ， HDA ）检测方法。根据葡糖醋杆菌16S基因序列（基因号为HQ677466.1 ）设计特异性引物，并优化反应体系中 UvrD解旋酶和T4 gp32 的浓度。通过HDA法直接检测酸乳中的葡糖醋杆菌，并确定其检出限，在建立的HDA体系中 对酸乳中多种菌株进行扩增和电泳检测，验证方法的特异性。结果表明：用HDA法检测酸乳中葡糖醋杆菌，得到 2 了与设计序列长度（101 bp）一致的基因片段，检出限为10 CFU/g ，反应体系中UvrD解旋酶和T4 gp32 的适宜添加 量分别为0.1 μg和5.0 μg。该方法用于检测酸乳中葡糖醋杆菌的灵敏度高、耗时短。 关键词：赖解旋酶恒温基因扩增法；酸乳；葡糖醋杆菌；检测 Detection of Gluconacetobacter in Yogurt by Helicase-Dependent Isothermal DNA Amplification 1 1,* 2 2 2 2 2 2, * TANG Xinyi , LIU Bo , ZHANG Tao , LI Yongyan , ZHANG Cuixia , WANG Zan , ZHANG Jingjing , ZHOU Wei (1. Beijing No.12 High School, Beijing 100071, China; 2. Hebei Key Laboratory of Food Safety, Hebei Food Inspection and Research Institute, Shijiazhuang 050071, China) Abstract: A helicase-dependent isothermal DNA amplification (HDA) assay was developed for the rapid and accurate detection of Gluconacetobacter in yogurt. A pair of specific oligonucleotide primers according to the 16S ribosomal RNA gene (GenBank accession number: HQ677466.1) of Gluconacetobacter was designed, and the optimal concentration of UvrD helicase and T4 gp32 in the reaction system were determined. The HAD method enabled direct detection of Gluconacetobacter in yogurt.