User:Jarle Pahr/Meat

Sequences for species identification:

For papers on species identification of meat products, see:

Authentication of species in meat products by genetic techniques: http://link.springer.com/article/10.1007%2Fs00217-010-1417-1?LI=true#page-1

A set of degenerate primers was designed for amplification and sequencing of the cytochrome b (cyt b) gene of several species:

MEAT F:  CGAGGCCTMTAYTAYGG
MEAT R:  ATTGAKCGTAGGATTGCGTA

PCR reactions were carried out in 50 uL volume with 100-300 ng DNA template. PCR program: "3 min at 95C, then 35 cycles (30 s at 95C, 30 s at 50C, and 30 s at 72C) and a final extension step of 72C for 3 min." Genomic DNA was extracted from 30 mg muscle tissue, following the protocol of Roger and Bendich (Roger SO, Bendich AJ (1988) Extraction of DNA from plant tissues. Plant Mol Biol Manual A6:1–10). For highly processed products, 150 mg sample was used. Sequencing was carried out using the same primers as for PCR. DNA amplification with the primers MEAT F/R gave an amplicon of 555 bp in all tested species.

Ilhak and Arslan. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique. Turk. J. Vet. Anim. Sci. 2007; 31(3): 159-163: http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf

The primers used by Ilhak and Arslan were designed based on Lahiff (2001). Mol Cell Probes. 2001 Feb;15(1):27-35. Species-specific PCR for the identification of ovine, porcine and chicken species in meta and bone meal (MBM).

PCR was performed in 50 uL reaction with Taq polymerase. An annealing temperature of 58 C was used for all primers. PCR program: 94 C 45 s, 58C 45 s and 72C 90 s. 30 cycles. Agarose gel electrophoresis was performed with a 1.5 % agarose gel, loaded with 15 uL PCR product and run at 100 V for 2 h.

Animal	Direction	Sequence	Position in reference sequence	Reference sequence accession
Bovine	FWD	GCCATATACTCTCCTTGGTGACA	8107/812	J01394
Bovine	REV	5’- GTAGGCTTGGGAATAGTACGA-	8377/8357
Sheep	FWD	5’- TTAAAGACTGAGAGCATGATA- 3’	71/91	AF039171
Sheep	REV	5’- ATGAAAGAGGCAAATAGATTTTCG- 3’	295/272
Porcine	FWD	5’- GCCTAAATCTCCCCTCAATGGTA- 3’	93/115	AF039170
Porcine	REV	5’- ATGAAAGAGGCAAATAGATTTTCG- 3’	304/281
Cat	FWD	5’- CATGCCTATCGAAACCTAACATAA- 3’	11101/11124	NC_001700
Cat	REV	5’- AAAGAAGCTGCAGGAGAGTGAGT- 3’	11373/11351
Dog	FWD	5’- GATGTGATCCGAGAAGGCACA- 3’	8821/8841	NC_002008
Dog	REV	5’- TTGTAATGAATAAGGCTTGAAG- 3’	9142/9121

Detection of Adulteration and Identification of Cat’s, Dog’s, Donkey’s and Horse’s Meat Using Species-Specific PCR and PCR-RFLP Techniques: http://www.ajbasweb.com/ajbas/2009/1716-1719.pdf

Abdel-Rahman et al. developed PCR and PCR-RFLP methods for identification of cat's, dog's, donkey's and horse's meat. The primers used were as follows:

Name	Length (bp)	Sequence	Tm (C) [calculated]	Tm (C) [Analytical]	GC (% / bp)	Comment
Cat’s SSR FWD		CTCATTCATCGATCTACCCA
Cat’s SSR REV		GTGAGTGTTAAAACTAGTACTAGAAGA
Dog’s SSR FWD		GGAGTATGCTTGATTCTACAG
Dog’s SSR REV		AGAAGTGGAATGAATGCC
Horse/Donkey SSR FWD		TTCTGCTCTGGGTGTGCTACTT				From Abdel-Rahman et al. 2009
Horse/Donkey SSR FWD		CTACTTCAGCCAGATCAGGC				From Abdel-Rahman et al. 2009
Donkey’s and Horse’s cytochrome-b FWD		CCATCCAACATCTCAGCATGATGAAA
Donkey’s and Horse’s cytochrome-b REV		GCCCCTCAGAATGATATTTGTCCTCA

SSR = Simple Sequence Repeat= RFLP = restriction fragment length polymorphism

Amplicon sizes from cat, dog and donkey were 672 and 808 bp respectively. The Horse/Donkey SSR primer and cytochrome-b primer pairs yielded amplicons of 221 and 359 bp, respectively. The annealing temperatures used for the four primer pairs were 52, 52, 55 and 58 C, respectively. PCR was carried out as follows: Initial denaturation: 94°C for 4 min. Amplification: 35 cycles, 94°C for 60s, annealing temperature at 52-58 for 60s, extensiona at 72°C for 60s. Final extension at 72°C for 10 min.

See also:

http://nxseq.bitesizebio.com/articles/neigh-need-for-ngs-for-meat-testing-in-the-mane-pcr-is-enough/

http://www.isag.us/index.asp?autotry=true&ULnotkn=true