Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Pol
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Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction Mohd Hazim Mohd Yusop & Shuhaimi Mustafa & Yaakob B. Che Man & Abdul Rahman Omar & Nur Fadhilah Khairil Mokhtar
Received: 17 January 2011 / Accepted: 2 June 2011 / Published online: 18 June 2011 # Springer Science+Business Media, LLC 2011
Abstract Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the Cq (quantification cycle) from 10 ng/μl porcine DNA is (18.70±0.12 to 19.08± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r2 =0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification. Keywords Real-time PCR . Molecular beacon . Pork . Cytochrome b gene
M. H. M. Yusop : S. Mustafa (*) : Y. B. Che Man : N. F. K. Mokhtar Halal Products Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia e-mail: [email protected] A. R. Omar Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Introduction Animal speciation in meat products is essential to detect adulteration or fraudulent substitution and to provide consumers accurate information about the product they purchase. The substitute materials are often similar to the main materials from biochemical and physical point of view and, hence, making their identification extremely difficult (Ghovvati et al. 2009). In relation to that, consumers demand strict safeguarding from falsely labeled meat products for a variety of economic, religious, and health reasons, which are enhanced by the recent crisis in the meat sector (Aida et al. 2007). According to the European Union labeling regulations, meat should be accurately labeled regarding their species content (European Commission 2001). The increasing demand for the transparency in the meat industry has provided the driving force in the development of methods for the analysis of meat products (Aida et al. 2005). Some of the meat adulteration cases also have been reported involving some other common species of animals, such as chicken, cow, sheep, goat, deer, and marine source (Ballin et al. 2009).
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