Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products

Abstract

Replacement of beef by buffalo and vice versa is frequent in global markets, but their authentication is challenging in processed foods due to the fragmentation of most biomarkers including DNA. The shortening of target sequences through use of two target sites might ameliorate assay reliability because it is highly unlikely that both targets will be lost during food processing. For the first time, we report a tetraplex polymerase chain reaction (PCR) assay targeting two different DNA regions in beef (106 and 120-bp) and buffalo (90 and 138-bp) mitochondrial genes to discriminate beef and buffalo in processed foods. All targets were stable under boiling, autoclaving and microwave cooking conditions. A survey in Malaysian markets revealed 71% beef curries contained buffalo but there was no buffalo in beef burgers. The assay detected down to 0.01ng DNA and 1% meat in admixed and burger products.

Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain

“…32 Literature search revealed that several qPCR methods are available for the identification and quantification of beef and buffalo 33 and beef and pork, 30,34 but no mqPCR systems have been documented for the simultaneous detection and quantification of cattle, buffalo, and porcine materials in the food chain. Recently, we have documented multiplex PCR 35 and PCR-RFLP 12 assays for the simultaneous identification of these materials in the food chain, but those methods are just limited to the qualitative detection; they cannot tell how much adulterants are present in the real-world specimens. Thus, the objective of the present study was to develop and validate a short-amplicon length tetraplex qPCR system that would allow both identification and quantification of cattle, buffalo and porcine derived materials in processed foods such as hotdogs, meatballs, and burgers, which are very popular on all continents.…”

Heptaplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Beef, Buffalo, Chicken, Cat, Dog, Pork, and Fish in Raw and Heat-Treated Food Products

“…To simulate an ordinary canning procedure and steam cooking process, we autoclaved the meat samples at 21 °C and 15 psi pressure for 20 min . The meat samples were exposed to microwave cooking, a fast and modern means of cooking, at 600 and 700 W for 30 min . The treated samples were then stored at −20 °C for DNA extraction.…”

A systematic review of DNA‐based methods in authentication of game and less common meat species

“…Technologically processed meat is at the forefront of meat product adulteration due to its complex and heterogeneous composition (Naveena et al., 2017), making it difficult to use simple, morphological, or organoleptic approaches to identify its composition. Meat species in the food chain have been previously identified based on protein markers (Montowska et al., 2014), lipid metabolites (Trivedi et al., 2016), and DNA nucleotides (Guntarti et al., 2017; Hossain, Ali, Hamid, Hossain, et al., 2017). DNA‐based technologies have the advantage of stability, uniformity, and polymorphism (Asing et al., 2016a; Druml et al., 2016) over other methods when detecting meat products.…”