Detection and discrimination of seven highly consumed meat species simultaneously in food products using heptaplex PCR-RFLP assay

Abstract

Food fraud is a global problem raising increased concerns during the past decades. Beef, buffalo, chicken, duck, goat, sheep, and pork are the heavily consumed meats bearing nutritional, economic and cultural/religious importance and are often found to be mutually adulterated in raw and processed states. To authenticate these species, we developed heptaplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using species-specific primer sets which amplified short-length amplicons (73–263 bp) targeting mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes. Target specificity was confirmed through cross-amplification reaction with 25 non-target species and PCR products were authenticated by restriction digestion with FatI, BfaI, and HPY188I enzymes followed by separation and visualization of fragments in an automated electrophoretic system. The detection limit was 0.5 % (w/w) meat in mixed matrices and food products. The assay was sufficiently stable under thermal treatments of boiling and autoclaving. A market survey on meat products revealed rampant substitution of beef with buffalo and integrity in chicken and porcine products. Given some advantageous features including short amplicons, exceptional stability and superior sensitivity, the developed assay could be conveniently used for discriminatory detection of target species in raw meat as well as processed meat products.