狗源性DNA检测试剂盒的研制与评价
Development and Evaluation of A Dog-derived DNA Test Kit
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摘要: 本研究旨在研制一种快速鉴定肉制品中狗源性成份的DNA检测试剂盒,并对此试剂盒做出方法学评价。以改良SDS-PK法提取狗肉及掺假肉品种(狐肉、貂肉)基因组DNA,选择具有种属特异性的线粒体细胞色素B基因作为鉴定靶基因,应用Primer5.0设计特异性引物,建立狗源性DNA检测方法,组装狗源性DNA检测试剂盒,并进行特异性、稳定性及重复性评价。结果表明,本试剂盒可有效检测肉制品中狗、狐、貂源性成分,DNA提取效率高,仅0.05 g肉样即可提取浓度高于100 ng/μL的基因组DNA,OD260/280处于1.80~2.00之间,最低检测限为10-4 ng/μL,所有检测步骤可在4.5 h内完成,操作简便,特异性高。试剂盒所采用试剂安全无毒,反复冻融20次后仍然有效,可于-20 ℃保存一年。随机抽取12份市售样品进行检测,有3份样本中存在狐、貂肉冒充狗肉现象,掺假率高达25%,该结果与长春食品药品检验中心检测结果一致。狗源性DNA检测试剂盒灵敏性高、特异性强,检测结果准确稳定,具有良好重复性,适用于狗肉快速定性检测。Abstract: To develop a DNA detection kit for rapid identification and make a methodological evaluation of the kit of dog-derived components in meat products,the genomic DNA of dog meat and adulterated meat(fox meat,clam meat)was extracted by modified SDS-PK method. The mitochondrial cytochrome B gene with species specificity was selected as the target gene for identification. Primer5.0 was used to design specific primers. Dog-derived DNA detection methods,assembly of dog-derived DNA detection kits,and specificity,stability and repeatability were evaluated. The results showed that the dog,fox and mink-derived ingredients in the meat products could be effectively detected by the kit,and DNA was efficiently extracted by the kit. Genomic DNA with a concentration higher than 100 ng/μL could be extracted from only 0.05 g of meat,OD260/280 was between 1.80~2.00,and the minimum detection limit was 10-4 ng/μL. All detection steps could be completed within 4.5 h. The advantages of simple operation and high specificity were provided by the kit. The reagents used in the kit were safe and non-toxic. It was still effective after repeated after freeze-thaw cycles for 20 times and it could be stored at-20 ℃ for one year.12 samples were randomly selected for testing. There were 3 samples with fox and mink meat posing as dog meat. The adulteration rate was as high as 25%,which was consistent with the test results of Changchun food and drug inspection center. The dog DNA detection kit had high sensitivity and specificity,accurate and stable detection results,good repeatability,and was suitable for rapid qualitative detection of dog meat.