meat identification

1. 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

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
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 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

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,

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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