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、1fg/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。
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.
作者
韦涛
黄继锦
黄惠琳
黄晓韵
谢耐珍
张璜
WEI Tao;HUANG Ji-Jin;HUANG Hui-Lin;HUANG Xiao-Yun;XIE Nai-Zhen;ZHANG Huang(Guangxi-ASEAN Food Inspection and Testing Center,Nanning 530022,China;Guangzhou Double Helix Gene Technology Co.,Ltd.,Guangzhou 510320,China)
出处
《食品安全质量检测学报》
CAS
北大核心
2021年第8期3060-3070,共11页
Journal of Food Safety and Quality
基金
广西重点研发计划项目(桂科AB18126084)。
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
dog-derived ingredients
meat adulteration identification
loop-mediated isothermal amplification technology
quantitative real-time PCR
meat products
