A Method for Authentication of Meat by PCR Amplification of Species-specific Markers of Mitochondrial Origin

Authenticity is a vital element of food quality and safety. Adulteration and improper description of food have been found for a long time. The adulterated food often enters the supply chain and threatens the feelings as well as the health of the people. In general, the reason behind the adulteration is the revenue maximization, either by using a low-cost ingredient to substitute a more expensive one or to remove the valued component (Ioannis and Nikolaos 2005; Waghray et al., 2011).
       
A significant section of the population does not prefer to consume the meat of a particular animal species due to religious and health concerns. Therefore, meat adulteration has issues relating to social, religious, economic and public health. Such circumstances demand the development of rapid detecting techniques for the authentication of species of meat.
       
Various techniques have been developed for authentication of meat which includes isoelectric focusing (King 1984), chemometric analysis (Arvanitoyannis and Van Houwelingen-Koukaliaroglou 2003),western blot (Chvez et al., 2008), ELISA (Martin et al., 1991) etc. These techniques are effective but with some limitations. Alternatively, PCR-based techniques have proven to be the most authenticated technique for the identification of animal species because of their accuracy, specificity, precision, and sensitivity (Hazra et al., 2017). Species-specific PCR involves amplifying the specific region of the gene by using specific primers that target a species (Kim et al., 2019). Mitochondrial markers are more efficient than nuclear markers in species identification and authentication as PCR of species-specific markers of mitochondrial origin is relatively rapid, accurate, sensitive and cost-effective as compared to other PCR based assays (Rastogi et al., 2007). Mitochondrial DNA is a circular, small, extra-chromosomal genome with a high mutation rate (Boore 1999). It comprises 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes and a non-coding region called D-loop.Among them, 12S ribosomal RNA (12S rRNA) gene (Girish et al., 2007), 16S ribosomal RNA (16S rRNA) gene (Karlsson and Holmlund 2007), Cytochrome b (Cytb) gene (Maede 2006; Panwar et al., 2015), and D- loop region (Sosa et al., 2000) have been extensively used in meat authentication.
       
In the present study, authentication of different raw meat viz. beef, carabeef, mutton, chevon, chicken, duck meat and dog meat as well as meat samples subjected to different processing temperatures was carried out by PCR of species-specific mitochondrial Cytb and D-loop markers.

Collection of meat samples
 
Samples of beef, carabeef, mutton, chevon, chicken, duck meat and dog meat were collected randomly from different locations of the North-Eastern region of India. The meat samples were subjected to heat treatment in hot water (80^oC) to have 75^oC core temperature. They were also cooked in steam to have the core temperature of 95^oC. The samples were also subjected to autoclaving at a temperature of 121^oC and 15lb pressure.
 
Extraction of genomic DNA
 
Total genomic DNA was extracted from raw and processed beef, carabeef, mutton, chevon, chicken, duck meat, and dog meat samples by phenol–chloroform–isoamyl alcohol method (Sambrook and Russell 2001). Briefly, the meat samples were mixed with 10 volume of lysis buffer containing 10 mMTris–HCl, (pH 8.0), 100 mM EDTA (pH 8.0), 0.5% SDS and 0.1 mg/mL of Proteinase K and incubated for 3 h at 50^oC. The mixture was treated with 50 µg/mL of RNase A for 1 h at 37^oC. It was then extracted with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1 vol/vol) and again with an equal volume of chloroform. The DNA was precipitated by ethanol and 1 M ammonium acetate solution. Finally, DNA was dissolved in TE buffer and used for further analysis. The DNA was also extracted from the samples treated at different temperatures. The quality of genomic DNA was checked in 0.8% agarose gel electrophoresis. The concentration of total DNA extracted from various sources was estimated at 260 nm and the purity of DNA was checked by taking the ratio of O.D. readings at 260 nm and 280 nm using a UV-Visible spectrophotometer (Cary 60 UV-Vis, Agilent, USA).
 
PCR amplification of mitochondrial Cytb and D-loop markers
 
The extracted DNA samples were subjected to PCR with species-specific mitochondrial Cytb and D-loop markers in a thermal cycler (ProFlex Base, Applied Biosystems, USA). The list of the species-specific primers is given in Table 1. The primers were synthesized by Integrated DNA Technologies (IDT, India). PCR amplification was carried out in 0.2 mL PCR tubes containing 10 μL of PCR master mix, 1 μL (10 pmol) each of forward and reverse primers, 50 ng of DNA. The volume was made up to 25 μL with nuclease-free water. The PCR conditions for each marker are given in Table 2.
 

 

 
Agarose gel electrophoresis and gel documentation
 
The PCR products were analyzed using agarose gel electrophoresis. Agarose gel of 1.5% was prepared and samples were loaded onto the gel. Along with the samples, a 100 bp DNA ladder was also run in agarose gel electrophoresis workstation (BioRad, USA). The PCR products were visualized and photographed after electrophoresis using a Gel Documentation system (Gel Doc EZ, BioRad, USA).

PCR assay, based on specific amplification of mitochondrial Cytb gene and D-loop region with species-specific primers, was developed for the detection of different meat. Species-specific mitochondrial Cytb gene successfully amplified the unique fragments for mutton, chevon and dog meat. Similarly, unique fragments for beef, carabeef, chicken and duck meat were also amplified by species-specific mitochondrial D-loop markers. The sizes of the amplified products were 126 bp for beef (Fig 1), 226 bp for carabeef (Fig 2), 254 bp for mutton (Fig 3), 453 bp for chevon (Fig 4), 256 bp for chicken (Fig5), 292 bp for duck meat (Fig 6) and 100 bp for dog meat (Fig 7). In recent times, meat authentication has been gaining importance because of the increasing fraudulent substitution of superior quality meat with that of inferior quality to earn commercial benefits. These markers were also tested in meat samples which were subjected to different cooking temperatures ranging from 75-121^oC and could be successfully amplified from all the samples. The size of the amplified products were the same as in raw meat (Fig 1-7). These markers were further validated by checking it for cross-amplification in other meats. The markers were amplified successfully the target species, whereas no amplification products were obtained with DNA from the non-target species (Fig 8-14). It was observed that all the markers used in this study were highly specific for the target species.
 

 

 

 

 

 

 

 

 

 

 

 

 

 

       
Mitochondrial Cytb gene and D-loop region were utilized to develop PCR-based methods for unambiguous identification of beef, carabeef, mutton, chevon, chicken, duck meat and dog meat. Although previous methods for meat authentication proved to be useful, however, with some drawbacks. While reproducibility of RAPD-PCR is a matter of concern due to the necessity of extremely rigid conditions, PCR-RFLP technique and sequencing of mitochondrial genes are expensive and time-consuming. These techniques require more analytical work and analysis of the results isquite challenging. Further, complexities involved in the interpretation of the results of PCR-RFLP make the procedure unfeasible (Ilhak and Arslan 2007). Spectroscopy methods combined with chemometrics analysis of protein of meat (Arvanitoyannis and Van Houwelingen- Koukaliaroglou 2003) is another reliable method for meat species authentication which has been used to authenticate the beef samples using mid-infrared spectroscopy with a chemometric technique of soft independent modeling of class analogy (SIMCA) analysis. Due to the denaturation of muscle proteins in high-temperature processing, these methods cannot be used for the detection of thermal processed or cooked meat samples.
       
Since mitochondrial Cytb gene and D-loop region have high sequence variability even between closely related species, therefore, it is possible to select the sequences specific to particular species. Moreover, mitochondrial DNA is maternally inherited which implies only one allele exists in an individual and thus no sequence ambiguities are to be expected from the presence of more than one allele (Unseld et al., 1995). These markers were used in raw meat as well as samples treated with different cooking temperatures, ranging from 75^oC to 121^oC and the results were consistent and reproducible across different samples. This indicated that the markers were stable at all these temperature ranges and did amplify the same as in the raw meat. Different thermal conditions did not affect the PCR amplification confirming the ability of the protocol to amplify the species-specific marker of mitochondrial origin. The markers used in this study showed no cross-reactivity with the non-target species and confirmed the absence of any cross-amplification indicating its’ specificity. The results confirmed the fact that the heat stability and large copy number of mitochondrial DNA in meat tissue contribute to the protection and survivability of the DNA fragments that are sufficient to be amplified by PCR (Girish et al., 2004). Large numbers of mitochondria are present in an average cell and each mitochondrion contains multiple copies of mtDNA. Besides, the compact circular shape of mtDNA makes it more stable than nuclear DNA and thus more suitable for DNA detection in small and damaged samples. Further, it was proved that mitochondrial markers were more efficient than nuclear markers in species identification and authentication purposes (Rastogi et al., 2007). Therefore, these characteristics make mitochondrial DNA ideal for the identification of species origin of fresh as well as cooked meat.

The results of this study indicated that the PCR of species-specific markers of mitochondrial origin can be a rapid, easy, accurate and economical method for authentication of different meat viz. beef, carabeef, mutton, chevon, chicken, duck meat and dog meat as compared to other methods. The markers used in this study could be applied in detecting raw as well as cooked meat samples. Further, this will be useful for effective control of adulteration of meat.

The research work mentioned in this article was carried out under the ICAR-LBS Young Scientist Award project on ‘Farm-to-Fork risk profiling of hazards associated with pork supply chain in India’ and the World Bank funded APART project. The authors wish to thank Indian Council of Agricultural Research and APART for the financial assistance to undertake this project.