Loop-Mediated Isothermal Amplification Method to Detect Animal-Derived Ingredients in Edible Vegetable Oil and Hogwash Oil
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Abstract:
A loop-mediated isothermal amplification (LAMP) method was established to detect the presences of animal-derived ingredients in edible vegetable oils and hogwash oils. Two outer primers (F3 and B3) and two inner primers (FIP and BIP) were designed based on the mitochondrial Cytb genes of pig, cattle, chicken, and duck. The inner primers FIP/BIP, betaine, Mg2+, and other reaction parameters were optimized, and the amplified products were detected with real-time fluorescence LAMP. The specificity and sensitivity of the method were evaluated. The results showed that the optimal conditions were 0.2 μmol/L outer primers (F3 and B3), 1.6 μmol/L inner primers (FIP and BIP), 0.8 μmol/L loop primers (FLP and BLP), 1 mol/L betaine, 6 mmol/L Mg2+, 1.6 mmol/L dNTPs, and a reaction temperature of 63 ℃. Under the optimal conditions, the best amplification was achieved for the abovementioned four animal-derived ingredients. With this improved LAMP approach, the animal-derived ingredients in mixed oils containing 1% of animal oils and 5% of hogwash oils could be detected. This method exhibits a very high specificity and sensitivity for pig-, cattle-, chicken-, and duck-derived ingredients, can be applied in the detection of animal-derived ingredients in oils, and provides a technical support for identification of oils and hogwash oils.
