Analytical MethodsPolymerase chain reaction assay for identification of chicken in meat and meat products
Introduction
The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness and unhealthy competition in meat market (Arslan et al., 2006, Mane et al., 2006). Consumer should be protected from these malicious practices of meat adulterations by quick, precise and specific identification of species present in meat and meat products. Various methods are employed for detection of species origin of meat. These include sensory evaluation to latest DNA based assays. DNA based assays are gaining popularity in meat species identification due to their stability at high temperature and conserved structure within all individual of the species (Calvo et al., 2001, Girish et al., 2004). Earlier, DNA based assay employed for identification of species origin of meat was DNA hybridisation (Baur, Teifel-Greiding, & Leibhardt, 1987) but nowadays that has been replaced by PCR assays.
The PCR assays are employed for identification of species origin of meat using random primers (Saez, Sanz, & Toldra, 2004) to amplify the non-targeted DNA, while universal primers are used (Girish et al., 2005, Verkaar et al., 2002) for amplification of targeted genomic and mitochondrial DNA followed by restrictive enzyme digestion to differentiate meat species. The results of RAPD–PCR are non-reproducible due to requirement of high stringent conditions (Koh, Lim, Chua, Chew, & Phang, 1998), while PCR–RFLP is more time consuming, require more analytical work and result interpretation is complex. The result interpretation is even more complex in admixture meat and meat products (Irfan-Ilhak & Arslan, 2007). The specific detection of species origin of meat by PCR using species-specific primers is relatively quick, precise, sensitive and cost effective as compared to other PCR based assay (Mane et al., 2007). This species-specific PCR assay was previously used by various workers for detection of various mammalian and poultry species in meat and meat products (Arslan et al., 2006, Meyer et al., 1994).
These PCR assays targets genomic as well as mitochondrial DNA for the purpose of meat species identification, even in cooked meat under different processing conditions. However, in the present study the mitochondrial DNA was used for meat species identification because of the maternal inheritance of mitochondria, normally only one allele exists in an individual and thus no sequence ambiguities are expected from the presence of more than one allele (Unseld, Beyermann, Brandt, & Hiesel, 1995). The variable regions of the mitochondrial gene are present in thousands of copies per cell (Greenwood & Paboo, 1999), which increases the probability of achieving a positive result even in severe DNA fragmentation due to intense processing conditions (Bellagamba, Moretti, Comincini, & Valfre, 2001). Keeping the above facts and considering future implications in the meat trade, the present study was planned with the overall objective to develop simple, quick, sensitive, precise, cost effective and applicable method for identification of chicken meat and meat products processed under different manufacturing conditions by PCR assay.
Section snippets
Meat and blood samples
The fresh meat samples of buffalo, sheep, goat, pig, chicken and duck were collected from the local municipal slaughterhouses. While the guinea fowl, turkey and quail samples were collected from central avian research institute. After collection, samples were kept at −20 °C till further processing. The blood samples were collected from specific breeds of species under investigation from different sources. The blood was collected in sterile 15 ml polypropylene tube containing 0.5 ml of 0.5 M
Results and discussion
Precise, authentic and rapid testing of animal origin foods is indispensable to avoid unfair market competition and protection of consumer from fraudulent practices of meat adulteration. So, the aim of the study was to develop and evaluate the species-specific PCR assay for identification of chicken species in meat and meat products processed under different manufacturing conditions. The species-specific primer pair was designed based on mitochondrial D-loop gene for amplification of about 442
Conclusions
The species-specific PCR assay was found to be precise, sensitive and rapid methods for routine analysis of meat species, even in admixed meat and meat products under different processing conditions. Thus, it can be concluded that the species-specific PCR assay can be useful tool for routine assessments of authenticity and quality of meat and meat products to protect the consumers from fraudulent practices of meat substitution.
Acknowledgement
The authors are thankful to Director, Indian Veterinary Research Institute, Izatnagar-243 122 (India) for providing necessary facilities for the present work.
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