A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

Abstract

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

A Fast and Reliable Real‐Time PCR Method for Detection of Ten Animal Species in Meat Products

“…Some methods are based on the DNA intercalating dye SYBR green to detect pork in poultry meat products (Soares and others ) and bovine and poultry in foodstuffs (Safdar and Junejo ). A few publications are focused on the use of EvaGreen dye to identify pork and horse meat (Sakalar and others ) and to detect beef and soybean in sausages (Safdar and Abasıyanık ), for example. To the best of our knowledge, this is the 1st study to use the real‐time PCR and melt curve analysis using an SYTO fluorescence dye to detect animal species in meat products.…”

“…Species adulteration in food is, typically, an intentional act for economic gain, using sources of low‐priced meats, such as pork and chicken, in high‐value meat products (Premanandh ). Pork, for example, is a potential source of replacement for beef and lamb due to the color and texture similarities (Sakalar and others ). The presence of undeclared species in food products may also be unintentional in some cases, due to negligence in production or cross‐contamination, when, for example, improperly cleaned equipment is used to process meat from different species (Cawthorn and others ).…”

Detecting mislabelling in meat products using PCR–FINS

“…Meat speciation is technically challenging, especially for extensively processed products. This challenge can be best met by DNA based techniques such as Southern Blotting with species-specific satellite DNA probes, Random Amplified Polymorphic DNA fingerprinting (RAPD), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), Multiplex PCR and PCR-FINS (Matsunaga et al 1999;Lin et al 2005;Aida et al 2005;Minarovic ˇet al 2010;Li et al 2011;Sakalar et al 2015).…”

Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Detection of Raccoon Dog in Meat Mixtures

“…Therefore, the development of a specific, accurate, and feasible technique through which raccoon dog meat can be detected is urgent. The identification of the species origin of meat is performed primarily through the standards of sensory evaluation and determination of proteins and oligonucleotides [3][4][5]. However, the histological and morphologic characteristics or speciesspecific epitopes might be altered or destroyed during meat processing and cooking.…”