肉中猪源性成分Real-time PCR定量检测技术  

Quantitative Detection Technology of Porcine-Derived Materials in Meat by Real-time PCR

作　　者：翟晓虎[1] 李翎旭 陈小竹 蒋怀德 贺卫华[1] 姚大伟[2] ZHAI XiaoHu;LI LingXu;CHEN XiaoZhu;JIANG HuaiDe;HE WeiHua;YAO DaWei(Jiangsu Agri-Animal Husbandry Vocational College,Taizhou 225300,Jiangsu;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095)

机构地区：[1]江苏农牧科技职业学院,江苏泰州225300 [2]南京农业大学动物医学院,南京210095

出　　处：《中国农业科学》2023年第1期156-164,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金青年基金(30471225);泰州市科技支撑计划社会发展(指导性)项目(TS202019)。

摘　　要：【目的】建立一种快速、准确的肉中猪源性成分定量检测方法。【方法】首先从GenBank数据库中筛选猪特异性的微卫星DNA,根据微卫星DNA核酸序列设计引物,对常见10种动物基因组DNA进行PCR扩增,通过有无扩增产物判断筛选的微卫星DNA对猪源性成分的特异性。然后根据微卫星DNA核酸序列,设计特异性引物和探针,建立猪源性成分Real-time PCR检测方法,采用双标准曲线分别对猪源性成分和总动物源性成分进行定量,计算猪源性成分的百分含量。【结果】筛选到猪特异性微卫星DNA(Accession EF172428),根据其序列设计的引物SEQ-sus2-F/R只能从猪基因组DNA中扩增出目的条带,其他动物的基因组均无目的条带扩增。建立的Real-time PCR检测方法灵敏度为0.02 ng/25μL反应体系。该方法能够准确检测出混合DNA样品中猪源性成分和混合肉样品中猪源性成分,百分误差分别约为1.32%和1.06%-7.12%。【结论】本研究利用Real-time PCR技术建立的定量猪源性成分的检测方法可以用来检测猪源性成分在混合样品中的百分含量。【Objective】The aim of this study was to develop a rapid and accurate quantitative method for identifying porcine-derived materials.【Method】Porcine microsatellites DNA were selected from GenBank nucleotide database. Primers specific for porcine were designed based on the sequences of microsatellite DNA. Genomic DNA from 10 kinds of common animals was amplified by PCR method. The specificity of selected microsatellite DNA to porcine-derived materials was judged by the amplification products.According to the microsatellite sequence, the specific primers and probes were designed to establish a Real-time PCR method for identifying porcine-derived materials. The double standard curve was used to quantify the porcine-derived materials and total animal-derived materials, respectively, and the percentage content of porcine-derived materials was calculated. 【Result】Porcine specific microsatellite DNA with the accession number EF172428 was selected. Only porcine DNA gave a fragment through PCR assay, while there was no amplification for other non-target animal species DNA. The limit of detection was 0.02 ng in a 25 μL reactive system using the Real-time PCR method. This method could accurately detect porcine-derived components in mixed DNA samples and mixed meat samples with 1.32% percent error and 1.06%-7.12% percent error, respectively. 【Conclusion】The quantitative detection method of porcine-derived materials by Real-time PCR in this research could be used to detect the percentage content of porcine-derived materials in mixed samples.

关 键 词：肉 猪 动物源性成分 Real-time PCR 定量 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]