They said this type of fraud was especially important for those who can't eat certain types of meats.
The single assay platform targeted two different gene sites in cows, pigs and buffaloes and six sets of primers specific to these species were designed.
It was sensitive enough to detect all the beef, buffalo and pork targets in raw and processed frankfurter products with as low as 0.1% adulteration.
Double target in single test
Currently, testing involves sampling DNA, amplifying the genetic material with the polymerase chain reaction (PCR) and looking for certain markers.
But existing methods often only search for one, long DNA sequence, which could break down during processing and lead to false results.
The multiplex polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay targeted two different gene sites - mitochondrial cytochrome b and ND5 genes.
All targets (73, 90, 106, 120, 138, and 146 basepairs) were stable under extreme boiling and autoclaving treatments.
Six pairs of primers (two of each species) were designed targeting cytb and ND5 genes of cow, buffalo, and pig species to develop a double gene targeted mPCR assay with short length of amplicons.
Species specificity of all targets was confirmed by cross-checking all primers against 27 non-target species.
Help prevent or reduce adulteration
PCR-RFLP assays can authenticate a product by restrictive digestion of the amplified PCR products using one or more restriction enzymes (REs).
“In this regard, multiplex PCR-RFLP (mPCR-RFLP) assay, especially the double gene targeting one with short amplicon targets, would be especially useful and trustworthy for the simultaneous detection of beef, buffalo, and pork products in various food products,” said the researchers.
“In this study, we have developed double gene targeted mPCR assay involving short length of the targets (73–146 bp) which are thermodynamically more stable than those of the longer targets.
“Because of the presence of double targets for each species, this novel assay could complement the detection of a missing target because it is highly unlikely that both gene sites would be lost under the states of decomposition.
“A confirmatory low-cost analytical test involving all three species definitely can help market regulation, preventing or at least reducing adulteration events to a great extent.”
The simplex PCR products of beef cytb and buffalo ND5 genes were digested with EciI and AluI restriction endonucleases, respectively. Beef ND5 and buffalo cytb products were digested with FatI.
Pork Pocytb and PoND5 PCR products were digested with CviKI-1 and FatI restriction endonucleases in a separate reaction tube of 25 μL reaction volume comprising 1 μg of unpurified PCR product, 1× digestion buffer supplied with the enzyme, 1U of each enzyme, and sterilized distilled water.
The authors reported a double gene site and short amplicon length (≤146 bp) mPCR-RFLP and tested it under raw, boiled (98 °C for 90 min), and autoclaved (121 °C and 15 psi pressure for 2.5 hours) atmospheres for identification of beef, buffalo, and pork in pure, admixed and frankfurter formulation.
They also screened 20 halal branded beef frankfurters in Malaysian markets.
All beef frankfurters were beef and buffalo positive; indicating all beef frankfurter products in Malaysia were buffalo adulterated.
The team also checked chicken and pork frankfurters, but none were beef and buffalo positive, adding this was probably because prices of beef and buffalo are higher than chicken and pork.
Source:J. Agric. Food Chem., 2016, 64 (32), pp 6343–6354
Online ahead of print, DOI: 10.1021/acs.jafc.6b02224
“Double Gene Targeting Multiplex Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Assay Discriminates Beef, Buffalo, and Pork Substitution in Frankfurter Products”
Authors: M. A. Motalib Hossain, Md. Eaqub Ali, Sharifah Bee Abd Hamid, Asing, Shuhaimi Mustafa Mohd Nasir Mohd Desa and I. S. M. Zaidul