环介导等温扩增技术在鸭源成分检测中的研究

Rapid Detection Technique of Duck-Derived Components in Beef and Sheep Products by LAMP

DOI：10.3969/j.issn.1673-1689.2019.08.018

中文关键词: 牛、羊肉制品 等温扩增技术 环介导等温扩增技术 可视化检测

英文关键词: bovine and mutton products,isothermal amplification techniques,loop-mediated isothermal amplification,visualization detection

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中文摘要:

为了建立肉产品中鸭源成分的现场快速检测技术，根据动物种间特异性的原则，筛选出一对品种特异的PCR引物，在此引物扩增片段的基础上，设计了环介导等温扩增（LAMP）引物序列，能特异检测鸭源成分。通过条件优化实验，发现在25 μL的扩增体系中，内外引物浓度比为1∶4，温度为63.5 ℃，MgSO4添加量为5 mM/μL，dNTPs添加量为1.75 mM/μL，Bst DNA聚合酶添加量为0.4 U/μL，甜菜碱添加量为0.25 μmol/μL，LAMP反应时间为30 min时，LAMP检测体系检测效果达到最优，鸭肉DNA的检测灵敏度可达到10 pg/μL。对牛羊肉中鸭源成分比例的检测灵敏度为0.000 1%。应用该方法对上海市闵行区市场中的牛羊肉产品进行抽样检测，结果从21份样品中成功检测出5份含鸭源成分的混合肉。本研究建立的LAMP检测方法灵敏、快捷，可以实现鸭源成分的现场速测，为肉制品市场的监管提供了一种新方法。

英文摘要:

A pair of specific PCR primers was selected based on the principle of animal species specificity in order to establish a duck meat products source components on-site rapid detection technology. The fragment was obtained by the specific primers only in duck samples. On the basis of this fragment，the ring mediated isothermal amplification（LAMP） primers were designed and the primers could detect duck source component specifically. By conditions optimization experiments，the most optimal amplification system for LAMP（25 ?滋L） as follow：concentration ratio of inside primer and outside primer was 1∶4，temperature was 63.5 ℃，MgSO4 was 5 mM/μL，dNTPs was 1.75 mM/μL，Bst DNA polymerase was 0.4 U/μL，betainewas 0.25 μmol/μL，the LAMP reaction time was 30 min. Under the optimized conditions，the detection sensitivity of duck DNA was 10 pg/uL. The detection sensitivity of component proportion for beef or mutton in the duck source was 0.000 1%. 21 samples from markets of Shanghai Minhang district were detected using this method and 5 samples contained duck source. The results showed LAMP was a sensitive rapid technology. It provides a new method to realize on-site detection of duck source components for meat products.

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