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

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 μmol/L inner primers(FIP and BIP),0.2 μmol/L outer primers(F3 and B3),6 mmol/L MgSO₄,1.6 mmol/L dNTP,10×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.