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)

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 cytochromeb
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,minimumof 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.