Identification of species (meat and blood samples) using nested-PCR analysis of mitochondrial DNA

Crocodile meat product is an alternative protein source. Although, crocodile meat is more expensive, its taste is similar to that of chicken and fish. The authentication of commercial meat species is important for consumer’s confidence. In this study, sensitive and specific method multiplex nested-PCR was applied to identify commercial meat species. Dried blood was used as an alternative DNA source for detection. The detection sensitivity was enhanced by primers specifically designed to encompass the mitochondrial Cytochrome b and NADH dehydrogenase 5/6 genes. The specificity and sensitivity of multiplex PCR system were tested. Different lengths of specific nested-PCR products were detected to be 350, 570, 750 and 1000 bp for chicken, pig, cow, and crocodile, respectively. The system allowed detection with as little as 5 nanogram of DNA from either meat or blood sample. Detection sensitivity of individual species was improved, enabling the detection of DNA with as little as 1 picogram. Cross reaction was not detected among the tested species. It was shown that the multiplex-PCR assay enhanced the sensitivity of routine species identification and allowed the use of blood as an alternative DNA source for detection.

[1]  W. Chaeychomsri,et al.  Freeze-dried crocodile blood production as food supplement , 2009 .

[2]  Ashok Kumar,et al.  Identification of chicken, duck, pigeon and pig meat by species-specific markers of mitochondrial origin. , 2009, Meat science.

[3]  A. Tiwari,et al.  Polymerase chain reaction assay for identification of chicken in meat and meat products. , 2009 .

[4]  F. Vogensen,et al.  Species determination - Can we detect and quantify meat adulteration? , 2009, Meat science.

[5]  S. Radu,et al.  Meat species identification and Halal authentication analysis using mitochondrial DNA. , 2009, Meat science.

[6]  A. Moussavi,et al.  Fraud identification in industrial meat products by multiplex PCR assay. , 2009 .

[7]  A. Gulluce,et al.  Identification of meat species by TaqMan-based real-time PCR assay. , 2009, Meat science.

[8]  M. Saito,et al.  Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification. , 2009, The Analyst.

[9]  Wentao Xu,et al.  A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species , 2009 .

[10]  S. Zhao,et al.  Rapid detection of bovine milk in yak milk using a polymerase chain reaction technique. , 2009, Journal of dairy science.

[11]  L. Hoffman,et al.  The yield and nutritional value of meat from African ungulates, camelidae, rodents, ratites and reptiles. , 2008, Meat science.

[12]  Z. Kesmen,et al.  PCR assay for the identification of animal species in cooked sausages. , 2007, Meat science.

[13]  E. Miyauchi,et al.  PCR Method of Detecting Pork in Foods for Verifying Allergen Labeling and for Identifying Hidden Pork Ingredients in Processed Foods , 2007, Bioscience, biotechnology, and biochemistry.

[14]  Thomas W Vickroy,et al.  Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[15]  F. M. Ghazali,et al.  Meat molecular detection: Sensitivity of polymerase chain reaction-restriction fragment length polymorphism in species differentiation of meat from animal origin , 2007 .

[16]  R. Lightowlers,et al.  Mitochondrial DNA clonality in the dock: can surveillance swing the case? , 2006, Trends in genetics : TIG.

[17]  S. C. Gerlach,et al.  Identification of animal species by protein radioimmunoassay of bone fragments and bloodstained stone tools. , 2006, Forensic science international.

[18]  J. Chisholm,et al.  The detection of horse and donkey using real-time PCR. , 2005, Meat science.

[19]  A. Puyet,et al.  Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction. , 2005, Analytical biochemistry.

[20]  Renping Wang,et al.  Identification of Chinese alligators (Alligator sinensis) meat by diagnostic PCR of the mitochondrial cytochrome b gene , 2005 .

[21]  A. Raha,et al.  Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. , 2005, Meat science.

[22]  P. Hebert,et al.  Identification of Birds through DNA Barcodes , 2004, PLoS biology.

[23]  F. Toldrá,et al.  PCR-based fingerprinting techniques for rapid detection of animal species in meat products. , 2004, Meat science.

[24]  J. Lenstra,et al.  Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA. , 2002, Meat science.

[25]  N. Huang,et al.  Cytochrome b gene for species identification of the conservation animals. , 2001, Forensic science international.

[26]  J. Boore.  Animal mitochondrial genomes. , 1999, Nucleic acids research.

[27]  S Muroya,et al.  A quick and simple method for the identification of meat species and meat products by PCR assay. , 1999, Meat science.

[28]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .