动物肌肉组织DNA的提取方法及实时荧光定量PCR检测  

The method of DNA extraction from animal muscle tissue and real- time PCR detection

作　　者：刘娜 赵新 陈锐 王成 朱珠 王永 兰青阔 

机构地区：[1]天津市农业质量标准与检测技术研究所,天津300381

出　　处：《食品工业科技》2016年第18期74-80,共7页Science and Technology of Food Industry

基　　金：天津市应用基础与前沿技术研究计划(14JCQNJC14800)资助

摘　　要：以生鲜羊肉、猪肉、鸡肉、牛肉、鸭肉为实验材料,采用SDS法、异硫氰酸胍法、试剂盒法提取动物肌肉组织基因组DNA,对提取的DNA进行琼脂糖凝胶电泳和实时荧光定量PCR检测。结果表明,试剂盒法提取的基因组DNA在浓度和纯度方面均优于SDS法和异硫氰酸胍法。通过对三种提取方法建立的标准曲线进行比较,回归系数(R2)按照质量排序为:试剂盒法≥异硫氰酸胍法>SDS法,扩增效率按照质量排序为:试剂盒法>异硫氰酸胍法>SDS法。三种提取方法的羊源性成分灵敏度检测,最低检出限均为80 pg/μL,但在低浓度检测时试剂盒法的相对标准偏差RSD为0.684%,小于异硫氰酸胍法0.734%和SDS法1.075%;猪源性成分灵敏度检测,SDS法的最低检出限为80 pg/μL,异硫氰酸胍法和试剂盒法的最低检出限均为16 pg/μL,但在低浓度检测时试剂盒法的相对标准偏差RSD为0.092%,小于异硫氰酸胍法4.640%;鸡源性成分灵敏度检测,SDS法和异硫氰酸胍法的最低检出限均为3.2 pg/μL,试剂盒法的最低检出限为640 fg/μL,明显高于SDS法和异硫氰酸胍法。综上所述,试剂盒法提取的基因组DNA更有利于运用实时荧光定量PCR技术进行食物掺假和物种鉴别工作。Genomic DNA of muscle tissues from fresh mutton, pork, chicken, beef, duck were extracted through SDS method,GuSCN method and kit method .The DNA was detected by agarosegel electrophoresis and real-time PCR method.Results indicated that the kit method improved better concentration and purity of DNA than SDS method and GuSCN method.By comparing the three extraction methods to establish standard curve,the sorted according to the quality of the regression coefficients： kit method ≥ GuSCN method 〉 SDS method.The amplification efficiency according to the sorting for quality was kit method ≥ GuSCN method 〉 SDS method.The detection sensitivity of sheep-derived ingredients sensitivity by three Extraction Methods,the minimum detection limits were 80 pg/μL, but in the low-concentration detecting the relative standard deviation（RSD） of the kit method 0.684% was less than GuSCN method 0.734% and SDS method 1.075%.The detection of pig-derived ingredients sensitivity,the minimum detection limit of SDS method was 80 pg/μL.The minimum detection limit of GuSCN method and kit method were 16 pg/μL,but in the low-concentration detecting the relative standard deviation （RSD）of the kit method 0.092% was less than GuSCN method 4.640%. The detection of chicken- derived ingredients sensitivity, the minimum detection limit of SDS method and GuSCN method were 3.2 pg/μL,the minimum detection limit of kit method was 640 fg/μL.Above all,the genomic DNA from kit method was more conducive to use the real-time PCR technique in food adulteration and species identification.

关 键 词：动物肌肉 SDS法 异硫氰酸胍法 试剂盒法 DNA 实时荧光定量PCR 

分 类 号：TS251.1[轻工技术与工程—农产品加工及贮藏工程]