两种致病性弧菌二重LAMP-熔解曲线检测方法的建立  

Development of a Multiplex LAMP-Dissociation Curve Method for the Detection of Two Pathogenic Vibrio Species

作　　者：姜侃 张水峰 张慧 汪新 刘鹏鹏 

机构地区：[1]浙江省质量检测科学研究院,浙江杭州310018

出　　处：《现代食品科技》2016年第10期246-251,238,共7页Modern Food Science and Technology

基　　金：浙江省优先主题重点社会发展项目(2009C13013);浙江省质监系统科研计划项目(20120101)

摘　　要：应用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP),建立了食品中产毒素性霍乱弧菌和副溶血性弧菌二重LAMP-熔解曲线快速检测方法。本方法针对产毒素性霍乱弧菌ctx A基因和副溶血性弧菌gyr B基因分别设计引物组进行二重LAMP扩增,并利用熔解曲线法分析扩增产物,从而判断DNA模板中所含目标菌。应用本方法对9株目标菌和17株非目标菌的检测结果与预期一致,并可通过熔解曲线的特征峰准确分析DNA模板中所含目标菌。对二重LAMP扩增产物的测序分析表明,扩增所得序列与目的基因序列吻合,从而进一步验证了该方法的特异性。经测试,本方法对两种目标菌的检测灵敏度均可达100 fg DNA/反应管。实验证明所建立的方法具有良好的特异性,并可为食品中两种致病性弧菌的快速检测提供一种重要技术手段。Loop-mediated isothermal amplification （LAMP） was applied to establish a rapid duplex LAMP-dissociation curve method for the simultaneous detection of toxigenic Hbrio cholerae and Hbrio parahaemolyticus in foods. The primers were designed according to the sequences of the ctxA gene of Vibrio cholerae and the gyrB gene of Vibrio parahaemolyticus, and the amplification products were analyzed by dissociation curves to determine the target bacteria using the DNA template. The results from the detection of nine target bacterial strains and 17 non-target bacterial strains were consistent with the expected results, and the target bacteria represented by the DNA template could be accurately analyzed using the characteristic peaks in the dissociation curve. DNA sequencing results of LAMP products indicated that the amplified sequences were also consistent with the target gene sequences, further confirming the specificity of this LAMP method. The experimental results showed that the detection limits for the two target bacteria could reach 100 fg DNA/tube. The results suggest that the established method has a high specificity, and can be used as an important technique for the rapid detection of two pathogenic Vibrio species in foods.

关 键 词：多重LAMP 熔解曲线 霍乱弧菌 副溶血性弧菌 食品 

分 类 号：TS207.4[轻工技术与工程—食品科学]