Evaluation of different DNA extraction methods for the detection of adulteration in raw and processed meat through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

摘要

Methods currently used for the identification of the species to confirm the origin of meat or white tissue samples have not been validated for identification of different classes of vertebrates used for meat production, such as fishes, mammals and birds. Here, we describe an improved method for extraction, detection and differentiation of meat species using a single set of primers-for mitochondrial cytochrome-b gene by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based procedure-to detect adulteration in meat. The work compared three different methods (viz., salt precipitation, silica column based extraction & chloroform-phenol extraction) for extracting total genomic DNA. Of the three methods, salt precipitation and silica column based extraction was found better in terms of extraction efficiency and ease of handling as compared to the standard method of chloroform-phenol extraction which is laborious. To confirm the effectiveness and specificity of the cytochrome-b gene fragment, we tested nine genomic DNA samples (extracted from different aquatic species as well as other meat samples) and obtained positive results. Using this method, adulteration of meat (aquatic or otherwise) up to 0.01 % in case of raw meat; and, minimum of 1 % adulteration in case of cooked and processed meat mixtures could be detected. In conclusion, specific PCR-RFLP method resulting in an amplicon of size 360 bp of a short segment of cytochrome-b gene seems to be a powerful technique for the identification of adulteration in raw, cooked and/or processed meat products because of its simplicity, specificity and sensitivity.