1. Meat Species Specification by Molecular Techniques Naveen Soni 1, S.S.Ahlawat 1, Jayanti Tokkas 2, Shalini Jain 3 and Hariom Yadav 4 1 Department of livestock Product Technology, LLRU VAS, Hisar, Haryana; 2 Department of Biochemistry, COBS & H, CCS HAU, Hisar, Haryana; 3 Department of Biochemistry, PGIMER, Chandigrah; 4 National Agri-Food Biotechnology Institute, Mohali, Punjab, India Correspondence: yadavhariom@gmail.comyadavhariom@gmail.com Naveen Soni 1, S.S.Ahlawat 1, Jayanti Tokkas 2, Shalini Jain 3 and Hariom Yadav 4 1 Department of livestock Product Technology, LLRU VAS, Hisar, Haryana; 2 Department of Biochemistry, COBS & H, CCS HAU, Hisar, Haryana; 3 Department of Biochemistry, PGIMER, Chandigrah; 4 National Agri-Food Biotechnology Institute, Mohali, Punjab, India Correspondence: yadavhariom@gmail.comyadavhariom@gmail.com

2. Meat species specification is an utmost important field of quality control management in meat industry. These practices are also helpful in implementation of prevention of cow slaughter acts of different states of India. Wild life conservation act. PFA acts of India and other similar acts of the world INTRODUCTION

3. Methods of Meat Identification  Physical techniques:  Colour  Texture  Odour  Presence of other body parts along with meat

4.  Anatomical techniques: The typical dental formulations Identification is on the basis of vertebrae Ribs number present on the carcass.  Histological techniques: Muscle fiber length Diameter Density Pattern of the muscle fibers

5.  Chemical techniques: Determination of fat in meat Determination of ash in edible bone meal  Biological techniques: Also known as Serological or Immunological methods. Precipitation test Complement fixation test (CFT) Enzyme-linked immunosorbent assay (ELISA) Radio immuno assay (RIA)

6. Molecular techniques  DNA based molecular techniques: 1. PCR based 2. Non PCR based

7. PCR based techniques  Species Identification by Specific PCR  Species Identification by PCR-RFLP(Polymerase chain reaction - Restriction fragment length polymorphism)  Species Identification by RAPD(random amplification of polymorphic DNA)  Species Identification by using FINS(Forensically Informative nucleotide sequencing)

8. What is PCR? It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993. PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.

10. PCR Amplification Exponential Amplification of template DNA

11. Species Identification by Specific PCR Two methods- 1. Specific PCR targeting nuclear DNA 2. Specific PCR targeting mitochondrial DNA

12. Specific-PCR targeting nuclear DNA  Detection of pork meat amplified Porcine Growth Hormone Gene (108 bp fragment) In heat treated meat products amplified the conserved region of 18S-ribosomal gene (137 bp fragment)

13. Agrose gel electrophorosis of 18S ribosomal gene.

14. Differentiation of Mammalian, Poultry and Fish meat mixture Targeted 18S r DNA gene to get specific PCR product of 293, 254 and 267 bp for Mammalian, Poultry and Fish meat respectively. Targeted of Growth hormone Gene in cattle and swine, and fragments of different length 130 bp for Cattle, 105 bp for Swine obtained.

15. Specific-PCR targeting mitochondrial DNA  As nuclear DNA, mt-DNA also used for designing species specific primer for meat identification. Cytochrome-b Gene primer: used for identification of beef and pork. Multiplex PCR using primer Cytochrome-b Gene were able to detect chevon, chicken, beef, mutton and horse meat respectively added to pork. Mitochondrial D-loop region species specific primers designed to detect pork meat in meat products.

16. In comparison to nuclear DNA, mt-DNA isolation is more easy due to the presence of multiple copies in a cell. The mt-DNA copies range from 100-10,000 per cell (except in egg and sperm cell). Hence, very small samples can be tested. In case of very old biological samples mtDNA analysis is used because mt-DNA easily isolated from samples like hair shafts, bones and teeth. The mt-DNA more stable and strong than the nuclear DNA. mt-DNA is protected from degradation, even when exposed to prolonged environmental conditions. Advantage of Specific-PCR targeting mitochondrial DNA

18. Species-specific amplification of mitochondrial D-loop region of sheep using newly designed primers: (lane M) 100 bp DNA ladder; (lane N TC ) no template control; (lane Ch) chicken; (lane P) pig; (lane G) goat; (lane S) sheep; (lane H) horse; (lane Cm) camel; (lane B) buffalo; (lane Ca) cattle

19. Species Identification by PCR RFLP (Polymerase chain reaction-Restriction fragment length polymorphism) PCR-RFLP involves PCR amplification of a gene followed by digestion with restriction enzymes. Meat spp.can be detected PCR amplification of DNA followed by species specific cleavage with a restriction enzyme. PCR-RFLP is a convenient, rapid, sensitive and versatile assey for meat spp.identification.

21. PCR-RFLP targeting nuclear DNA Mutton and chevon can be differentiated by PCR-RFLP analysis of satellite-I DNA as Apa-I restriction enzyme has site in sheep but goat be not. Restriction profile of melanocortnin gene (MCR) has been used for differentiated of Hanoow meat from Holstein and Angus meats. Differentiated of meat from Taurine cattle, Zebu cattle, Banteng, Bison, Wsient, Water buffalo and African buffalo by PCR-RFLP process on Centrometric Satellite DNA, digested with EcoR-II, BamH-I and Pst-I.

22. (a) EcoRI1 and (b) BamH1 restriction profiles obtained from PCR-RFLP analysis of Centrometric Satellite DNA in cooked meat from nine animal species. Lane numbers 1=molecular marker of 1ooo bp; 2= Taurine cattle; 3= Zebu cattle; 4= Banteng; 5=buffalo; 6= Bison; 7= Wsient; 8= Water buffalow; 9=African buffalow; 10=Indian cattle.

23. PCR-RFLP targeting mitochondrial DNA Cytochrome-b Gene is most common used target for PCR- RFLP. PCR amplification of 359 bp of Cytochrome-b Gene fragment and cut with Alu-I, Rsa-I, Taq-I and HinF-I to identified Cattle, buffalo, horse, pig, wild boar, sheep, goat and chicken, meat. PCR amplification of 981 bp Cytochrome-b Gene fragment and cut with Alu-Iand Nco-I to identified fallow deer, red deer, roe deer and chinkara.

24. Species Identification by Randomly Amplified Polymorphic DNA (RAPD) It is a type of PCR reaction, but the segments of DNA that are amplified are random. We use arbitrary primer to use amplify DNA fragments in different spp.and clear distinct patterns with high level of polymorphism were detected between spp.while fewer polymorphism found with in spp.

25. RAPD PROFILES OF MEATS FROM VARIOUS SPECIES The sequence of the 10-base random primer used was ACGACCCACG M, marker; 1(, bear; 2, rabbit; 3, dog; 4, cat; 5, donkey; 6, horse; 7, wild swine; 8, pig; 9, camel;10, sheep; 11, goat; 12, beef.

26. Species Identification by using Forensically Informative nucleotide sequencing (FINS) Forensically informative nucleotide sequencing (FINS), a technique that combines DNA sequencing and phylogenetic analysis. It is used to identify samples based on informative nucleotide sequences. PCR amplification and sequencing of conserved gene is one of the first techniques for meat spp.identification. Mitochondrial DNA is highly conserved, gene on it Cytochrome-b and 12S-r RNA used for meat spp.identification.

28. Advantage of PCR based techniques We can be detected the wide variety meat samples. Fresh or processed meat can be easily detected. Much reliable. Very small amount of adulteration (up to 1%) can be easily identified.

29. Species identification by hybridization Dot-blots hybridization technique has been applied to the detection of species-specific DNA fragments in the cooked meats of chicken, pig, goat, sheep, and beef. The probes, biotin-labeled chromosomal DNA fragments, were hybridized to the sample DNA on nylon membranes. The species of the meats were identified at 100 ng/dot of the sample DNA. Non PCR based

30.  Advantages - Simple and quick Easy to perform Can be perform everywhere  Disadvantages Costly Heat sensitive Species specific probe is required

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