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, India
Correspondence: yadavhariom@gmail.com
 INTRODUCTION

•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

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 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)
 What is PCR?
PCR is an exponentially progressing synthesis of
the defined target DNA sequences in vitro.

It was invented in 1983 by Dr. Kary Mullis, for which

he received the Nobel Prize in Chemistry in 1993.
 PCR Amplification
Exponential Amplification of template DNA
Species Identification by Specific PCR

Two methods-
1. Specific PCR targeting nuclear DNA
2. Specific PCR targeting mitochondrial DNA
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)
Agrose gel electrophorosis of 18S ribosomal gene.
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.
 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.
 Advantage of Specific-PCR targeting
mitochondrial DNA

 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.
 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)

 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 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.
(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
 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)

 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
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.
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
 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.
 Non PCR based

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.
Advantages-
 Simple and quick
 Easy to perform
 Can be perform everywhere

 Disadvantages
 Costly
 Heat sensitive
 Species specific probe is required
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