环介导等温扩增与qPCR用于检测肉制品中狗源性成分的比较

Comparison of loop-mediated isothermal amplification and qPCR used for the detection of the dog-derived ingredients in meat products

中文关键词:  狗源性成分  肉类掺假鉴定  环介导等温扩增技术  实时荧光PCR  肉制品

英文关键词:dog-derived ingredients  meat adulteration identification  loop-mediated isothermal amplification technology  quantitative real-time PCR  meat products

中文摘要:

      目的 建立基于实时荧光PCR和环介导等温扩增检测肉制品中狗源性成分的检测方法并比较2种方法的检测效能。方法 针对狗线粒体CYTB基因保守序列, 采用Primer Explorer Version 4软件设计环介导等温扩增引物, Primer Express 3.0.1软件设计实时荧光PCR引物及探针。提取狗肉基因组DNA作为阳性模板, 黄牛肉等20种基因组DNA作为阴性对照, 分析扩增特异性; 用含靶序列的人工质粒确定检测灵敏度及人工掺肉样确定最低检出限; 用市售肉制品分析检出率。结果 对于肉制品中狗源性成分检测, 实时荧光PCR和环介导等温扩增检测均有较好的特异性, 人工质粒分析灵敏度分别为0.1、1 fg/μL。对10种市售狗肉制品的狗源性成分均检出阳性、10份不含狗肉制品均检出阴性, qPCR法和环介导等温扩增法的检测结果符合率均为100%。结论 qPCR法和环介导等温扩增法在检测肉及肉制品中的狗源性成分时, 在灵敏度、检出限和准确度上具有相当的效能。

英文摘要:

      Objective To establish detection methods based on real-time fluorescent PCR and loop-mediated isothermal amplification for detecting dog-derived ingredients in meat products and compare the detection performances of the two methods. Methods Aiming at the conservative sequence of dog mitochondrial CYTB gene, the loop-mediated isothermal amplification primers were designed using Primer Explorer Version 4 software and the real-time fluorescent PCR primers and probes were designed using Primer Express 3.0.1 software. The genomic DNA of dog meat was extracted as the positive template, and 20 kinds of genomic DNA such as yellow beef were used as the negative control to analyze the amplification specificity. The artificial plasmid containing target sequences were used to determine the detection sensitivity and the artificial mixed meat samples were used to determine the minimum detection limit. The detection rate was analyzed with commercial meat products. Results For the detection of dog-derived ingredients in meat products, real-time fluorescent PCR and loop-mediated isothermal amplification both had good specificity, and the analytical sensitivities of artificial plasmids were 0.1 and 1 fg/μL, respectively. The dog-derived ingredients of 10 commercial dog meat products were positive, and those of 10 products without dog meat were negative. The coincidence rates of detection results by qPCR method and loop-mediated isothermal amplification method were both 100%. Conclusion The qPCR method and the loop-mediated isothermal amplification method are quite efficient in detecting dog-derived ingredients in meat and meat products with respect to sensitivity, detection limit and accuracy.