Beef, Buffalo and Pork Detection In Food Chain Using Double Gene-targeted Multiplex PCR Assay

Introduction: Identification of animal species in food products has become an important and routine work under the supervision framework of most countries to prevent diseases transmitted by meat and bone meal as well as meat adulteration by mixing materials of different animal origins.  Specifically, adulteration of porcine, bovine and buffalo material are of serious religious, health and economic concerns. Multiplex polymerase chain reaction (PCR) assays are greatly promising for the screening of multiple species targets in a single assay platform, saving analytical cost and time.

Purpose: The aim of the study was to develop double gene targeted short amplicon-length multiplex PCR assay for the confirm detection and differentiation of beef, buffalo and pork in raw and processed food products. Compare to a single target assay, targeting double gene sites must enhance the assay reliability through a complementation approach since it is unlikely that both gene targets will be missing under the food processing treatments. 

Methods: Six different sets of primers, two for the each of beef, buffalo and pig, were developed targeting multicopy mitochondrial cytochrome b (cytb) and ND5 gene after the screening of specificity through bioinformatic software, mismatch analysis, 3D plotting and phylogenetic analysis.  The final specificity was confirmed through PCR analysis against 20 different meat species followed by separation using automated capillary electrophoresis.

Results: All the six targets of length between 73 and 146 bp appeared both in the gel-image and electrpherogram both under raw and processed states of pure and complex food matrices of burger, meatball and frankfurter formulations.

Significance: It was the first report of a double gene targeted multiplex PCR assay for the confirmed detection of beef, buffalo and pork in food chain. The method would cut down the detection cost of the said species in food forensic or any archaeological investigation by at least three folds.