动物源性成分实时等温扩增检测方法的建立

摘要

Objective To establish a rapid real-time loop-mediated isothermal amplification (LAMP) method for rapid detection of animal derived materials. Methods The specific LAMP primers of chicken, duck, cattle and sheep were designed according to the specific mitochondrial cytb, COX3, 12S rRNA and cytb genes, respectively. Target DNA was amplified by real-time LAMP with fluorescent dye SYTO-9 within 45 min at an isothermal temperature of 63 ℃. The reaction results were judged by detecting the fluorescence intensity in real-time, and real-time monitoring was carried out. Results The detection limits of LAMP assay were 1 pg genomic DNA for chicken and duck and 10 pg for cattle and sheep.Thedetectionlimitsforadulteratedmeatproductscouldreach0.01％forchicken,duck,cattleandsheep. Conclusion This method for rapid detection of meat products has the advantages of simplicity, low cost, real-time monitoring, high sensitivity and specificity, and is suitable for rapid on-site detection of supervision departments.%目的 建立一种实时荧光环介导等温扩增法(loop-mediated isothermal amplification,LAMP)快速检测动物源性成分的分析方法.方法 根据鸡、鸭、牛、羊物种cytb、COX3、12S rRNA、cytb特异性基因分别设计LAMP引物,反应时加入荧光染料SYTO-9,在63℃条件下反应45 min,通过实时检测荧光强度判断反应结果,并进行实时监测.结果 实时荧光LAMP方法对鸡、鸭源性成分的检测灵敏度为1 pg,对牛、羊源性成分的检测灵敏度为10 pg.对熟肉制品中掺杂肉检测时,掺杂鸡肉、鸭肉、牛肉和羊肉的检出限为0.01％.结论 该方法可用于快速检测肉制品中动物源性成分,具有操作简单、无需贵重仪器、反应过程可实时监控、灵敏度高和特异性强等优点,适合于基层监管部门的现场快速检测.