可视化环介导等温扩增技术检测鸡鸭源性成分

Visual detection of chicken and duck derived components by loop-mediated isothermal amplification method

中文关键词:  鸡源性成分  鸭源性成分  环介导等温扩增  可视化检测

英文关键词:chicken derived component  duck derived component  loop-mediated isothermal amplification  visual detection

中文摘要:

      目的 建立生肉及熟肉制品中鸡、鸭源性成分的特异性基因LAMP可视化快速检测方法。方法 根据鸡鸭物种特异性cytb、COX3基因的6个特异性区域设计LAMP引物, 在反应前加入羟基萘酚蓝(HNB), 在63 ℃条件下反应45 min; 反应结果可以根据颜色变化直接判断, 阳性为天蓝色, 阴性为紫罗兰色。并对30个肉制品样品进行可视化LAMP和PCR方法平行检测。结果 LAMP方法的最低检出限为1 pg, 特异性良好, 同时对肉制品中目标物检测的检出限可达到0.01%。结论 该方法用于肉制品中鸡鸭源性成分的快速检测, 具有操作简单、无需贵重仪器、反应结果肉眼可观察、灵敏度高和特异性强等特点, 适合于基层监管部门进行肉制品现场快速检测。

英文摘要:

      Objective To establish a visual and rapid detection technique for chicken and duck derived components by loop-mediated isothermal amplification (LAMP). Methods The specific LAMP primers of chicken and duck were designed according to the specific mitochondrial cytb and COX3 genes, respectively. Target DNA was amplified and visualized by hydroxyl naphthol blue (HNB) dye within 45 min at an isothermal temperature of 63 ℃ and the results were determined by the color change of HNB. Meanwhile, thirty samples were detected by LAMP and PCR. Results The detection limit of the LAMP assay was about 1 pg genomic DNA and had good levels of specificity. It was found that the LAMP assays allowed the detection of as little as 0.01% chicken or duck derived components in the meat mixtures. Conclusion The established method provides a useful tool for the rapid and sensitive detection of meat species, and it has the advantages of simple operation, no need for expensive instruments, observing the reaction results by the naked eye, high sensitivity and specificity, which is suitable for on-site rapid detection of meat products for grass-roots supervision departments.