Meat Species Specification by Molecular Techniques

Naveen Soni1, S.S.Ahlawat1, Jayanti Tokkas2, Shalini Jain3 and Hariom Yadav4

1 Department of livestock Product Technology, LLRU VAS, Hisar, Haryana; 2Department of Biochemistry, COBS & H, CCS HAU, Hisar, Haryana; 3Department of Biochemistry, PGIMER, Chandigrah; 4National Agri-Food Biotechnology Institute, Mohali, Punjab, IndiaCorrespondence: yadavhariom@gmail.com

•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

Methods of Meat Identification

Physical techniques: Colour Texture Odour Presence of other body parts along with meat

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

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)

Molecular techniques DNA based molecular techniques:

1.PCR based

2.Non PCR based

PCR based techniquesSpecies Identification by Specific PCRSpecies 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)

What is PCR?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.

PCR AmplificationPCR AmplificationExponential Amplification of template DNA

Species Identification by Specific PCRSpecies Identification by Specific PCR

Two methods-1.Specific PCR targeting nuclear DNA2.Specific PCR targeting mitochondrial DNA

Specific-PCR targeting nuclear DNA Specific-PCR targeting nuclear DNA

Detection of pork meatamplified Porcine Growth Hormone Gene

(108 bp fragment)

In heat treated meat products amplified the conserved region of 18S-ribosomal gene (137 bp fragment)

Agrose gel electrophorosis of 18S ribosomal gene.

Differentiation of Mammalian, Poultry and Fish meat mixtureDifferentiation 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.

Specific-PCR targeting mitochondrial DNASpecific-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.

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

Advantage of Specific-PCR targeting mitochondrial DNA

 Species-specific amplification of mitochondrial D-loop region of sheep using newly designed primers: (lane M) 100 bp DNA ladder; (lane NTC) 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

Species Identification by PCR RFLP(Polymerase chain reaction-Restriction fragment length polymorphism)

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.

PCR-RFLP targeting nuclear DNAPCR-RFLP targeting nuclear DNAMutton 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.

(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.

PCR-RFLP targeting mitochondrial DNA

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.

Species Identification by Randomly Amplified Polymorphic DNA (RAPD)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.

RAPD PROFILES OF MEATS FROM VARIOUS SPECIES The sequence of the 10-base random primer

used was ACGACCCACGM, 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.

Species Identification by using Forensically Informative

nucleotide sequencing (FINS)

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.

Advantage of PCR based techniques Advantage of PCR based techniquesWe 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.

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 Non PCR based

Advantages-Simple and quickEasy to performCan be perform everywhere

DisadvantagesCostlyHeat sensitiveSpecies specific probe is required

References References• Zimmermann, A., Hemmer, W., Liniger, M., Lüthy, J. and Pauli, U. (1998). A

sensitive detection method for genetically modified MaisGard TM corn using a nested PCR-system. LWT-Food Science and Technology 31: 664-667.

• Jankiewicz, A., Broll, H. and Zagon, J. (1999). The official method for the detection of genetically modified soybeans (German Food Act LMBG §35); a semi-quantitative study of sensitivity limits with glyphosate-tolerant soybeans (Roundup Ready) and insect-resistant maize (Maximizer). European Food Research and Technology 209: 77-82.

• Vollenhofer, S., Burg, K., Schmidt, J. and Kroath, H. (1999). Genetically modified organisms in food - screening and specific detection by polymerase chain reaction. Journal of Agricultural and Food Chemistry 47: 5038-5043.

• Berdal, K.G. and Holst-Jensen, A. (2001). Roundup Ready soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses. European Food Research and Technology 213: 432-438.

• Anklam, E., Gadani, F., Heinze, P., Pijnenburg, H. and Van Den Eede, G. (2002). Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. European Food Research and Technology 214: 3-26.

• James, D., Schmidt, A.M., Wall, E., Green, M. and Masri, S. (2003). Reliable detection of genetically modified maize, soybean, and canola by multiplex PCR a nalysis. Journal of Agricultural and Food Chemistry 51: 5829-5834

• . O lsen, J.E., Aabo, S., Hill, W., Notemans, S., Wernars, K., Granum, P.E., Popovic, T., Rasmussen, H.N. and Olsvik, O. (1995). Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. International Journal of Food Microbiology 28: 1-78

• . Hill, W.E. (1996). The polymerase chain reaction - applications for the detection of food-borne pathogens. Critical Reviews in Food Science and Nutrition 36: 123-173.

• Wang, R.-F., Cao, W.-W. and Cerniglia, C.F. (1997). A universal protocol for PCR detection of 13 species of food-borne pathogens in foods. Journal of Applied Microbiology 83: 727-736.

• Scheu, P.M., Berghof, K. and Stahl, U. (1998). Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction. Food Microbiology 15: 13-31.

• Chikuni, K., Tabata, T., Kosugiyama, M., Monna, M. and Saito, M. (1994). Polymerase chain reaction assay for detection of sheep and goat meats. Meat Science 37: 337-345.