Targeting double genes in multiplex PCR for discriminating bovine, buffalo and porcine materials in food chain

Abstract

Beef, buffalo and pork are the major meat of economic, religious and health concern. Current methods to authenticate these materials in food chain are based on mainly single gene targets which are susceptible to break down by food processing treatments. We, for the first time, described here a double gene targeting short-amplicon length multiplex polymerase chain reaction assay for discriminating bovine, buffalo and porcine materials in a single assay platform. The advantage of the assay is evidenced in terms of fidelity, cost and time since it is highly unlikely that two different targets would be missing even in a decomposed specimen. Detection of multiple targets in a single assay definitely saves analytical cost and time. Mitochondrial cytochrome b (cytb) and ND5 genes were targeted and six different targets (length: 90-146 bp), two for each of cow (120 and 106bp), buffalo (90 and 138bp) and pig (73 and 146bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The detection limit was 0.02 ng DNA under pure states and 0.1% meat in binary mixtures and meatball products. Screening of Malaysian meatball products revealed all beef products were buffalo positive in which 35% were totally replaced. In contrast, all pork products were found uncontaminated from beef and buffalo.

Species – specific PCR test for the quick recognition of equine tissue in raw and processed beef meat mixtures

“…The current extraction technique is less complicated and needs less technical requirements in comparison with one beforehand depicted by Di Pinto et al (2005) and Hossain et al (2017). The PCR products showed a high specificity of the PCR approach in connection to the outcomes acquired by Di Pinto et al (2005).…”

“…The capability to recognize questionable species in meat items is critical not only for economic, health, moral and religious reasons, but also to guarantee fair trade and commitment with related laws (Nakyinsige et al, 2012;Abbas et al, 2018). Most indicative strategies used to identify meat have relied upon the revealing of species-particular proteins or DNA (Ballin et al, 2009;Hossain et al, 2017). Today, DNA is considered to be the most appropriate particle for species identifying and distinguishing evidence in foods (Singh & Neelam, 2011).…”

Progress and challenges associated with halal authentication of consumer packaged goods

“…The limit of detection of the assay is comparable (0.1%) to that in other published reports. The advantage of the assay is its ability to complement potential missing targets, as it amplifies two different genes simultaneously …”

“…The advantage of the assay is its ability to complement potential missing targets, as it amplifies two different genes simultaneously. 28 Halal authentication is not limited to porcine detection alone. In general, products from animals with canine teeth or fangs, such as dog, cat, monkey and rat, are also considered non-halal according to Islamic laws.…”

PCR-RFLP identification of meat from red deer, sika deer, roe deer, fallow deer, mouflon, wild boar, hare and cattle

“…A variety of different molecular techniques can be employed for the determination of species origin, including that of game meat. These methods include PCR using species-specific primers (Ha et al 2006;Amaral et al 2014;Amaral et al 2015;Hossain et al 2017), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Pfeiffer et al 2004;Guan et al 2018;Jiang et al 2018), real-time PCR (Santos et al 2012;Druml et al 2015;Kaltenbrunner et al 2018), nucleotide sequencing (Barbuto et al 2010;Abdigini et al 2015) and the random amplified polymorphic DNA (RAPD) technique (Martinez and Yman 1998;El-Jaafari et al 2008).…”