环介导等温扩增技术快速检测大西洋鲑的研究

为探究环等温扩增技术(LAMP)在鲑科鱼类物种鉴定方面的应用,以大西洋鲑为研究对象,针对其线粒体CR区段设计特异性的LAMP引物,并通过单因素试验和正交试验研究反应体系中d NTP Mix、Mg~(2+)、甜菜碱终浓度对LAMP扩增结果的影响。结果表明,大西洋鲑LAMP扩增的最优条件为:引物SS-CR-F3/SS-CR-B3各0.2 m M,SS-CR-FIP/SS-CR-BIP各1.6 m M,dNTP Mix 1.4 m M,Mg~(2+)6 m M,甜菜碱0.8M,Bst DNA聚合酶8 U,扩增温度为65℃,反应时间60 min。在此条件下LAMP扩增产物电泳检测特异性强、稳定性好,对大西洋鲑DNA的检出限达0.01 ng·μL~(-1),并可对混合样本进行检测。本研究结果为大西洋鲑快速物种鉴定提供了一种重要技术手段,该技术可应用于鲑科鱼类物种鉴别的日常检验检疫和市场监管工作中。

Loop-mediated Isothermal Amplification Method (LAMP) for the Rapid Identification of Salmo salar

To investigate the application of loop-mediated isothermal amplification (LAMP) method for salmon species identification,a set of species-specific LAMP primers was designed based on the CR fragment of the mitochondrial DNA of Salmo salar.The effects of different concentrations of dNTP Mix,Mg2 + and Betaine on LAMP results were analyzed by the single factor and orthogonal experiments.The optimal conditions were determined as follows:0.2 mM of primer SS-CR-F3 and primer SS-CR-B,1.6 mM of primer SS-CR-FIP and primer SS-CR-BIP,1.4 mM of dNTP Mix,6.0 mM of Mg2+,0.8 M of Betaine,8 U of Bst DNA polymerase in 25 μL reaction mixture.The LAMP reactions were carried out at 65℃,for 60 minutes.Under these optimized conditions,the LAMP method displayed high degrees of specificity,sensitivity and stability,with the detection limit of 0.01ng.μL-1.This method could detect S.salar either in single or mixed salmon specimen.This study provided an important technical means for rapid identification of S.salar,which could be utilized in routine inspection & quarantine and market supervision of salmon species.