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目的建立一种能够快速区分出绵羊肉中掺入的狐狸源性成分的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。方法针对狐狸线粒体Cox I基因分别设计两条外引物和两条内引物,优化LAMP反应条件后并进行特异性和灵敏性试验,对绵羊肉中狐狸源性成分进行检测。结果在0.2μmol/L外引物(F3和B3)、1.6μmol/L内引物(FIP和BIP)、0.5 mol/L甜菜碱、0.4 mmol/L d NTP、4 mmol/L Mg SO_4等参数的优化条件下,可检测出掺假率为1.0%的羊肉制品,即最低检测浓度为2×10~(-4)ng/μl。结论本文所建立的LAMP法特异性强、灵敏度高,能有效地对绵羊肉中狐狸源性成分进行检测,可作为羊肉掺假的一种快速检测方法。
Objective To establish a loop-mediated isothermal amplification (LAMP) method for the rapid identification of fox-derived components incorporated into sheep meat. Methods Two exogenous primers and two primers were designed respectively for the mitochondrial Cox I gene in fox. The LAMP reaction conditions were optimized and the specificity and sensitivity tests were performed to detect the fox-derived components in sheep meat. Results Optimization of parameters such as 0.2 μmol / L external primer (F3 and B3), 1.6 μmol / L internal primer (FIP and BIP), 0.5 mol / L betaine, 0.4 mmol / L dNTP and 4 mmol / L MgSO 4 Under the conditions, can detect adulteration rate of 1.0% of mutton products, the minimum detection concentration of 2 × 10 ~ (-4) ng / μl. Conclusion The LAMP method established in this paper has high specificity and high sensitivity, which can effectively detect fox-derived components in sheep meat and can be used as a rapid detection method for lamb adulteration.