环介导恒温扩增快速检测植物油中掺假地沟油方法的建立

Establishment of rapid method for detecting adulterated hogwash oil in vegetable oil by loop-mediated isothermal amplification

以植物油中不应存在动物源性成分为鉴定依据,建立食用植物油中掺假地沟油的环介导恒温扩增(Loopmediated isothermal amplification,LAMP)快速检测方法,以动物线粒体DNA为检测靶标,根据脊椎动物线粒体基因组高度保守序列设计了两条外引物(F3、B3)和两条内引物(FIP、BIP),采用LAMP实时荧光法和染色法对扩增产物进行比较分析,对甜菜碱和Bst DNA聚合酶(Bst DNA polymerase)量进行了优化,并进一步评价方法的特异性、灵敏度和稳定性。结果表明,在1.6μmol/L内引物(FIP和BIP),0.2μmol/L外引物(F3和B3),6 mmol/L Mg SO_4,1.6 mmol/L d NTP,10×Thermo pol Buffer,8 U Bst DNA聚合酶,扩增温度为65℃,扩增时间为1 h的优化条件下,能够一次同时扩增出牛、羊、猪和鸡等脊椎动物DNA序列,检出植物油中最低含量为0.01 ng/μL的动物源性DNA,优化后的LAMP方法特异性好,仅能扩增出动物源性DNA序列、重复性好、稳定性高,可用于油脂中动物源性成分的检测,为油脂鉴伪和地沟油的鉴别提供一种稳定、高效、适应性强的现场快速检测技术。

To establish a method of loop-mediated isothermal amplification for detecting hogwash oil in edible vegetable oil, the animal mitochondrial DNA was targeted to detect the animal exogenous components which were not any vegetable oil ingredient. Two outer primers（ F3 and B3 ）and two inner primers（ FIP and BIP）were designed based on the highly conserved sequences in the vertebrate mitochondrial genome. The amplified products were compared with real time fluorescence loop- mediated isothermal amplification（LAMP） and staining method, betaine and Bst DNA polymerase amount were optimized.The specificity, sensitivity and stability of the method were evaluated.The results showed that the optimized condition was 1.6 Ixmol/L inner primers（ FIP and BIP） ,0.2 Ixmol/L outer primers（ F3 and B3 ） ,6 mmol/L MgSO4,1.6 mmol/L dNTP, 10 x Thermo pol Buffer, a 8 U Bst DNA polymerase, amplified at 65 ℃ for 1 hour.Using the LAMP, DNA of bovine, sheep, pig and chicken amplified in one reaction,and the lowest content in vegetable oil for animal derived was 0.01 ng/μL.The optimized LAMP method develops a good specificity and could only amplify the animal derived DNA sequence with good repeatability and high stability, suitable for rapid detecting of animal derived components in grease.LAMP provides a stable, efficient and adaptable rapid field detection of hogwash oil.