TaqMan实时荧光PCR法定量检测生肉中猪源性成分的建立  

Establishment of quantitative Taq Man real-time PCR targeting the swine-derived in raw meat

作　　者：徐琼[1] 张奕南[1] 顾文佳[1] 张清平[1] 胡雪莲[1] 赵燕[1] 曲勤凤[1] 

机构地区：[1]上海市质量监督检验技术研究院,上海200233

出　　处：《食品科技》2016年第2期309-313,共5页Food Science and Technology

基　　金：上海市质量技术监督局科研项目(2014-13)

摘　　要：目的：建立基于Taq Man实时荧光PCR技术的猪源性成分的定量检测方法。方法：选择朊蛋白基因为猪特异性基因和线粒体12S r RNA为内参基因,使用△Ct法进行相对定量。通过对其他种类的肉源DNA进行扩增,以验证方法的特异性;对含有1%猪肉的混合肉DNA进行系列稀释,确定检出限,并对自制模拟掺假肉进行猪源性掺假的测定,对该方法进行验证。结果：所用的引物具有较好的特异性,当模板浓度为50 ng/μL时对猪源性扩增的平均Ct值为22.73,而其他种类的肉源进行扩增时均未能检测到扩增信号,检出限为0.25%（w/w）;检测结果在猪肉含量为1%~100%（w/w）范围内具有良好的线性关系,相关系数R2=0.9955。内部验证结果表明,根据标准曲线得到的盲样检测结果同样品真实含量具有较高的一致性,变异系数在0.1270%~1.2044%之间。结论：所建立的Taq Man实时定量PCR法可用于生肉中猪源性成分的定量检测。Objective：A rapid quantitative detection method based on real-time PCR was established to detect the pork＇s mass percentage in raw meat.Methods：Porcine prion protein gene as porcinespecific primer and the mitochondrial 12 S r RNA as endogenous control were select to conduct relative quantification through △Ct method.The specificity of the established method was evaluated by direct DNA amplification with various meat samples,while the limit of detection was tested by serially diluted DNA which was extracted from the mixture containing 1% pork.To validate the proposed real-time PCR methodology,binary mixtures prepard with known amounts of swine in swine-mutton meat mixtures were analysed as blind samples.Results：Analysis of data showed that the Ct value from 50 ng/μL swine DNA is 22.73.Meanwhile,the other samples exhibited negative result.The limit of quantification is 0.25%（w/w）for swine in swine-mutton.Analysis of percentage of pork in meat mixtures showed the assay can determine 1%~100% contaminated swine,high linear regression（R2=0.9955）.The in–house validation using samples evidenced a high reproducibility of the methodology（coefficient of variation from 0.1270%~1.2044%）.Conclusion：The Taq Man real-time PCR methodology established by this study provides a reliable means for quantitative detection of adulteration in raw meat swine-derived components.

关 键 词：实时荧光PCR 生肉制品 猪源性成分 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]