该研究建立肉类食品中牛肉PCR-核酸试纸条快速鉴定方法,并开发快速检测试剂盒。对国家标准中牛的特异性引物进行标记,研发牛肉DNA快速提取试剂和基因检测试剂,采用核酸试纸条进行可视化检测;应用分子克隆及测序技术,克隆牛肉标准品;并对试剂的特异性、重现性、稳定性及灵敏度进行考察。提取基因组DNA的方法简单、快速,DNA的完整性、浓度及纯度均非常好;退火温度59 ℃、在20个循环时试纸条检测结果最特异,牛肉正品均出现2条条带,易混品及空白对照均出现1条条带。克隆测序后的牛肉DNA序列与牛线粒体DNA特异指纹区段序列同源性100%。自主研发的试剂盒特异性、重现性、稳定性良好,模板DNA最低检出量为1 pg/μL。该研究建立的PCR-核酸试纸条方法特异性强、灵敏、准确、简便、快速;研制的试剂盒操作简便快速、结果可视稳定,适用于实地监测。
The authenticity and safety of meat directly affect the health of consumers. A rapid identification method for beef in meat food using PCR-nucleic acid test strip and a rapid detection kit was developed in this paper. First, the cattle specific primers in GB were labeled, and the beef DNA rapid extraction reagents and beef gene detection reagents were developed, and nucleic acid test strips were used for visual detection. Then, molecular cloning and sequencing technologies were applied to clone standard beef DNA products. The specificity, reproducibility, stability and sensitivity of the reagents were investigated next. The method for genomic DNA extracting was simple and rapid, the integrity, concentration, and purity of DNA were excellent. The test strip showed the most specific results when annealing at 59 ℃ for 20 cycles. Two strips appeared in all the authentic beef samples, and one strip appeared in the confusing beef samples and the blank control samples. The sequence of the cloned beef DNA was 100% homologous to the specific fingerprint sequence of bovine mt DNA. The self-developed kit had good specificity, reproducibility and stability, the minimum detectable amount of template DNA was 1 pg/μL. The PCR-nucleic acid test strip method established in this study is specific, sensitive, accurate, simple and rapid. The developed kit is easy to operate, and the results are visible and stable, which is suitable for field monitoring.
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