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Recent Developments in Meat Species Speciation-a review

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 Singh et al, 2014/J. Livestock Sci. 5: 49-64

Recent Developments in Meat Species Speciation-a

review
V.P. Singh*, V. Pathak, N.K. Nayak, Akhilesh, K. Verma and P.
Umaraw

Department of Livestock Products Technology, U.P. Pt. Deen Dayal Upadhyay Veterinary University and
Go Anusandhan Sansthan, Mathura, U.P. India 281001
*Corresponding author: Mob.: +91-9412190865 Email: [email protected]

Journal of Livestock Science (ISSN online 2277-6214) 5:49-64

Received on 18/06/2014; Accepted on 17 /07/2014

Abstract
Meat species speciation is important to validate the quality and quantity of meat and meat
products. It helps in prevention of adulteration of inferior quality meat into superior quality
which is in practice since long back. The adulteration in the meat trade is a vulnerable issue and
sometime creates serious medico-legal and vetero-legal complications. So handling of meat trade
with authenticity is prime concern in meat species speciation. For this purpose numerous
techniques right from traditional methods to most modern techniques are being used. The
selection of right technique for particular meat identification is dependent on the need of test and
condition of meat used. The recent sophisticated techniques are able to identify even traces of the
meat added in the meat. Some techniques are also capable of identification of deteriorated meat
mixed with other meats.

Keywords: Meat, speciation, PCR, RFLP, RAPD, real time PCR

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Introduction

Meat is a highly nutritious commodity liked by most of the consumers. The

variety and quality of meat and its delicacy is dependant on the meat type. The variation in the
value of meat of various species is also dependant on local choice of the consumers and also
on nutritional status of the meat. So to earn more money from the meat business various types
of adulterations are very common. In other words, act of adding inferior quality meat with
superior one is known as fraudulent substitution. It is a common practice in many countries
of the world. Some common types of adulterations in meat business are mixing of horse
meat for beef in UK and Ireland, beef for kangaroo meat in Australia, cat for chicken or
rabbit meat, goat for mutton, mutton for venison, dog and cat meat for chevon etc.
The basic purposes of conducting meat species identification are now very much relevant to
ensure the quality and authenticity of the meat. The other purposes of conducting these
techniques includes quality control management in meat industry, food safety and human health,
conservation of laws, safeguard the religious sentiments, consumers satisfaction, fair trade,
economic importance, vetro-legal solution etc.
So to find out the meat species mixed with other meat, various types of methods are in use.
They are started from simpler techniques based on morphology to the sophisticated
techniques in which gene based technologies are used. Some common techniques adopted for
meat species speciation are physical techniques (differentiation in colour, consistency, odour,
marbling, presence of other body parts along with meat etc.), anatomical techniques (the typical
dental formulations, identification on the basis of vertebrae, ribs number present on the carcass
etc.), histological techniques (muscle fiber diameter, muscle fiber density, pattern of the muscle
fibers etc.), chemical techniques (determination of carotene, glycogen , refractive index, iodine
number etc.), biological techniques based on serological or immunological
phenomenon (precipitation test, complement fixation test (CFT), enzyme-linked
immunosorbent assay (ELISA), electrophoresis techniques etc.). Now a day various molecular
techniques are also in use which are the variables of polymerase chain reactions.

Biological or serological or immunological techniques

Ring precipitation test

It is a qualitative evaluation test in which antigen antibody reaction takes place and
at the point of interaction between antigens and antibodies a ring forms in case of positive test
for a particular meat. This test is also having some drawbacks like it is not a suitable
method for identification of mea species from heat treated meat. It also sometimes gives false
+ve results and formed ring diffused shortly.

Double Immunodiffusion Test

DID is also based on the same principles as ring precipitation test because
antigen and antibodies reaction takes place in both of the techniques. The basic difference
is in use of compliments to holds the bands for longer period of time and to enhance the
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visibility of the bands. In this technique known antiserum is used to test the mixture of meat or
meat samples. A band is forms at the point of interaction which can better visualize in the
presence of suitable compliments. It is suitable test for both qualitative and quantitative
assessment of meat adulteration. This technique is suitable for detection of meat adulteration
upto 5%. Meat cooked at 80°C for 10 min can easily be identified by this technology. The time
requires for performing the test is 2-3 days. However, test sometime gives false +ve result in
closely related species.

Overnight Rapid Identification Test

There are various types of test kits are available to identify the meat species from the
mixture of meat. The test alongwith their principles and utility is given in table 1.

Table 1 List of Overnight Rapid Identification Test (adopted from Jones and Patterson, 1985)

Test Principle Species identification

ORBIT Blank+ Precast Agar+ Overnight-PPT Beef
PROFIT Blank+ Precast Agar+ Overnight-PPT Poultry
MULTI-SIFT Blank+ Precast Agar+ Overnight-PPT Beef, Pork, Poultry, sheep,
Horse and deer meat
Dot Blot Antigen+ nitrocellulose or cynogen bromide activated Beef, Pork, Poultry, sheep,
Techniques nitrocellulose containing antibodies Horse & deer meat

Enzyme Linked Immunosorbent Assay (ELISA)

ELISA is a most common method used now a day in various purposes. It is
rapid and highly sensitive test for meat species speciation and results can be obtained within
2-3hrs. It is well suited technique for larger number of samples because numerous samples can
be handled at a time. It is a good technique for closely related species identification and
adulteration upto 2% can be easily detected. Pressure cooked meat at 133°C for 20 min. can
be identified by this technique. Various versions of ELISA are now a day used in the
techniques i.e. Indirect/competitive/sandwitch etc. (Patterson and Spencer, 1985).

Electrophoresis techniques
In this technique, separation of proteins takes place by their differential migration
through supportive medium under influence of electric field (Kim and Shelef, 1986). Thus
the protein bands resolved can be visualized by enzymological, chemical and immunological
means. This technique has good reproducibility and resolution. The common techniques
used are Polyacrylamide Agar Gel Electrophoresis (PAGE) used for identification of beef,
pork, chicken and turkey (fresh and frozen), Sodium Dodecyl Sulphate
Polyacrylamide Agar Gel Electrophoresis (SDS PAGE) used for beef, mutton, venison,
rabbit meat (raw/cooked) etc. Counter Immuno-electrophoris is another version of
electrophoresis used for the purpose. It is a type of immune-diffusion test in which alkaline
gel causes electro-osmosis. This is a suitable technique for detection of 1:300 dilutions
(Sherikar et al., 1988). It is rapid and more sensitive test for meat species identifications.

Isoelectric Focusing

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In IEC, migration of protein is in pH gradient principle is utilized. Species

specific bands forms which can be identified on the basis of location, density and area of bands.
This technique can be utilized for identification of fresh as well as cooked meat upto100°C.
However, IEC is not a suitable method for closely related species and frozen meat (Skarpeid et
al., 1998). For better visualization of whole muscles, coomassie blue, can be utilized while
phosphoglucomutase is suitable for identification of low levels of buffalo, pig or horse meat
in beef. The other added benefits in identification of low levels of kangaroo or horse meat in
beef can be achieved by using adenylate kinase and phosphor gluconate dehydrogenase
(PGD) for diffrentiation of mutton with chevon (King, 1984).

Chromatographic techniques
There are various types of chromatographic techniques are utilized for identification of
meat species. Cation exchange chromatography and high performance liquid chromatography are
the most common types of chromatographic techniques used for the purpose. In cation exchange
chromatography separation of haemoglobin followed by filtration with cellulose acetate paper is
done. The final step in this technique is diode array detection at 416 nm. By the use of
characteristics peak patterns of cation exchange chromatography species of meat can be specified
(Ashoor et al., 1998). By the use of High Performance Liquid Chromatography muscle
samples from beef, veal, lamb, pork and turkey can be compared and identify. This
method should provide a rapid method for detection of meat adulteration or for separation
and purification of muscle proteins (Toorop et al., 1997).

Molecular techniques

Most of the molecular techniques can be applied in meat species speciation but
most common technique is polymerase chain reaction (PCR). There are various variants of
PCR are available for this purpose.

Polymerase Chain Reaction (PCR)

PCR is a rapid technique in which multiple copies of specific piece of DNA
sequences in vitro can be obtained. It is a highly selective and specific test to find out the
species of meat in amixture of meat sample. It is a highly sensitive technique in which even a
single copy sequence from a single cell sample can be found out. It is qualitative test and
quality of the mixture and easily determined. These methods can be applied on closely
related meat species. PCR techniques are also capable for differentiation of meat from male
and female. The other benefits of PCR over other conventional methods include detection of
wide variety of meat samples. Fresh or processed meat can be easily detected by this
technique. It is much reliable and very small amount of adulteration (up to 1%) can be easily
identified.
In PCR techniques for meat speciation genetic markers are used. They may be nuclear
gene or mitochondrial gene markers. Among nuclear markers; Growth hormone gene (Brodmann
and Moor, 2003), Actin gene (Hopwood et al., 1999) and Melanocortin receptor 1 (MC1R) gene
(Fajardo et al., 2008a) are common while among mitochondrial gene used for this
purpose includes Cytochrome -b (Maede, 2006; Pfeiffer et al.,2004), 12S and 16S
ribosomal RNA subunits (Girish et al., 2007; Karlsson and Holmlund, 2007) and

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Displacement loop region (D- loop) (Krkoska et al., 2003; Montiel-Sosa et al., 2000). On
comparison of both these genes it can say that mitochondrial gene are more convenient and
applicable because mt-DNA isolation is more easy due to the presence of multiple copies in
a cell, mt-DNA copies range from 100-10,000 per cell (except in egg and sperm cell) hence
very small samples can be tested. These markers are also capable of detecting very old
biological samples. Another reason for its preference includes more stability of mt-DNA and
strong ness in comparison to nuclear DNA. mt-DNA is protected from degradation, even
when exposed to prolonged environmental conditions.

PCR sequencing
In this technique sequencing of a particular gene is carried out to know the nature of gene
responsible for particular meat species specificity. The work in this regard carried out is
tabulated in table 2.

Table 2 Work carried out on PCR Sequencing Technology for meat speciation
Workers Meat species speciation Technology adopted
Chikuni et al. Red deer species, as well as A 646 base pair (bp) fragment of the
(1994) some birds like quail, song mitochondrial cytochrome b gene
thrush and sparrow
Brodmann et Red deer, By sequencing the PCR products
al.(2001) fallow deer, roe deer achieved from a conserved 428
and chamois bp region of the mitochondrial
cytochrome b gene.
Wong et al. (2008) Snake meats to 355 bp cytochrome b sequence
enforce wildlife conservation
programs
Colombo et al. Meat samples suspected of Sequenced a 282 bp amplicon from the
(2004) containing chamois mitochondrial cytochrome b gene

Li et al. (2006) Cervid species By sequence analysis of 405 bp and

387 bp amplicons generated from
the mitochondrial cytochrome
b and 12S rRNA genes, respectively.
Kitano et al. Mammals, birds, Based on conserved regions using
(2007) reptiles, amphibians and primers designed to amplify small
fish fragments (from 100 to 244 bp)
on the mitochondrial 12S and
16S rRNA genes.
La Neve et Red deer, roe deer, pyrenean PCR-sequencing and capillary
al.(2008) ibex and chamois, cattle, electrophoresis techniques
sheep and goat targeting a 232 bp amplicon of the
mitochondrial cytochrome b gene
Girish et al. Quail, guinea fowl, ostrich and Targeting a 456 bp fragment from the
(2009) emu meat mitochondrial 12S rRNA gene
Lee et al. (2009) By-products PCR-sequencing of the
like elephant ivory mitochondrial cytochrome b gene
Hsieh et al. (2003) Horns from PCR-sequencing of the
rhinoceros species mitochondrial cytochrome b gene

Matsunaga et al. Meats from species Targeting nuclear markers,

(1998) and kangaroo, crocodile or buffalo genes like 18S rRNA or the diglyceride
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Venkatachalapathy acyl transferase1 (DGAT1) have been

et al. (2008) sequenced

DNA barcoding
Using the barcoding technology various scientists tried to find out the meat species. A list of
work carried out on this aspect is summarized in table 3.

Table 3 DNA barcoding meat speciation

Workers Meat species Technology adopted
speciation
Hebert et al. (2003), Various domestic DNA barcoding targets a small standardized fragment
Kitano et al. (2007) and wild of 650 bp on the mitochondrial
and Ferri et cytochrome oxidase I (COI) gene that is PCR
al.(2009) species amplified and sequenced to produce reference
sequences or “DNA barcodes”, which act as
molecular identification tags for each species
profiled.
Holmes et al. (2009) Shark and ray By DNA barcode analysis
species

Species specific PCR

Species specific PCR is a unique technique used to find out the specific meat
species from the mixture of meat samples. There are two types of the techniques are generally
used such as Specific PCR targeting nuclear DNA and Specific PCR targeting mitochondrial
DNA. The work carried out by several workers on this aspect included in table 4.

Table 4 PCR using species-specific primers for meat speciation

Species Genetic marker Specific PCR products Références

Ostrich and Emu Cytochrome b 543 and 229 bp Colombo et al. (2000)
Cervid species (Ceylon spotted deer, Ceylon Cytochrome b 450 bp Rajapaksha et al. (2002)
hog deer, Ceylon sambhur and barking deer)
Buffalo Cytochrome b 242 bp Rajapaksha et al. (2003)
Tiger Cytochrome b 408 bp Wan and Fang (2003)
Camel Cytochrome b 208 bp Chen et al. (2005)
Deer, cattle, sheep, goat and ruminants 12S and 16S 104, 99, 108, 105 and 191 Ha et al. (2006)
rRNA bp
Ostrich and emu Cytochrome b 543 and 229 bp Colombo et al. (2000)
Cervid species (Ceylon spotted deer, Ceylon Cytochrome b 450 bp Rajapaksha et al. (2002)
hog deer, Ceylon sambhur and barking deer)
Red deer, roe deer and fallow deer 12S rRNA 175, 169 and 175 bp Fajardo et al.(2007)
Pheasant, quail, guinea fowl, chicken, turkey, Cytochrome b 164, 187, 192, 133, 71, 95 Stirtzel et al.(2007)
duck and goose and 237 bp
Red deer, cattle, sheep, goat, domestic pig, Cytochrome b From 89 to 362 bp Tobe and Linacre (2008)
horse, donkey, cat, dog, fox, guinea pig,
hedgehog, badger, harvest mouse, house
mouse, rat, rabbit and human
Guinea fowl, chicken, duck, and turkey Cytochrome b 186, 188, 189 and 186 bp Nau et al. (2009)

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Pigeon, chicken, duck, and domestic pig Cytochrome b and 401, 256, 292 and 835 bp Haunshi et al. (2009)
D-loop region
Snake species (Indian rockrat snake and Indian 16S rRNA 380, 265 and 165 bp Dubey et al. (2009)
cobra)
Cetacean species 12S rRNA 172 and 49 bp Shinoda et al. (2009)
Quail, pheasant, partridge and guinea fowl 12S rRNA 129, 113, 141 and 130 bp Rojas et al. (2009b)

Quail, pheasant, partridge, guinea fowl, D-loop 96, 100, 104, 106, Rojas et al., (2010a)
pigeon, Eurasian woodcock and song thrush 147, 127, and 154 bp

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

polymorphism)
PCR-RFLP technology involves PCR amplification of a gene followed by digestion with
restriction enzymes. In this technology there are different types of enzymes are used in nuclear and
mitochondrial gene markers. In this technique meat species can be detected by PCR amplification of
DNA followed by species specific cleavage with a restriction enzyme. It is a convenient, rapid, sensitive and
versatile assay for meat species identification (Verma et al. 2013). Number of workers carried out the work
on this aspect list of some of them is given in table 5.
Table 5 Work carried out on PCR-RFLP for meat speciation
Species Enzymes Genetic marker (bp) Références
Red deer, roe deer, moose, antelope, chamois, AflIII, AluI, AseI, CfoI, DraI, Cytochrome b (359 bp) Meyer et al. (1995)
mouflon, wild boar, kangaroo, buffalo, cattle, sheep, DraIII, EcoRI, HaeIII, HindI,
goat, domestic pig, horse, chicken & turkey HindII, HinfI, MboI, MboII,
PstI, RsaI, SalI, SspI, TaqI,
Tru9I, XbaI
Red deer, sika deer, cattle, sheep, goat and BamHI, EcoRI, ScaI Cytochrome b (194bp) Matsunaga et al.
domestic pig (1998)
Red deer, fallow deer, roe deer, bison and hare AluI, NcoI Cytochrome b (981bp) Zimmer mann
et al. (1998)
Red deer, fallow deer, moose, antelope, gazelle, AluI, AseI, BamHI, Cytochrome b (464bp) Wolf et al. (1999)
wildebeest, chamois, pyrenean ibex, kangaroo, HaeIII, HincII, HinfI, MseI,
buffalo, cattle, sheep, goat and hare NlaIII, RsaI, SspI, TaqI
Red deer,kangaroo, buffalo, horse, cattle, sheep, HaeIII, HinfI Cytochrome b (359 bp) Partis et al. (2000)
goat, domestic pig, emu, duck, chicken, turkey,
rabbit, crocodile, barramundi, cat, dog, human,
salmon, tuna, Nile perch and John dory
Wild boar and domestic pig AvaII D-loop region (531bp) Montiel-Sosa et al.
(2000)
Wild boar and domestic pig Tsp509I D-loop region (531 bp) Krkoska et al.
(2003)
Red deer, roe deer, wild boar, horse, cattle, goat, AluI, HinfI, MboI, PalI Cytochrome b (359 bp) Pascoal et al.
sheep, domestic pig, partridge, ostrich, duck, (2004)
chicken, turkey and rabbit
Red deer, roe deer, cattle, sheep and goat Tsp509I Cytochrome b (195 bp) Pfeiffer et al.
(2004)
Buffalo, cattle, sheep and goat AluI, ApoI, BspTI, HhaI 12S rRNA (456 bp) Girish et al. (2005)
Red deer, fallow deer, roe deer, cattle, sheep and goat ApoI, BslI, MboII, MseI 12S rRNA (720 bp) Fajardo et al.
(2006)
Cervids, bovines, porcines, equines and birds AluI, HaeIII, HinfI, Cytochrome b Maede (2006)
MboI, PstI, RsaI, TaI, XbaI (359e218bp)
Wildebeest, zebra, gazelle, impala, buffalo, RsaI D-loop region Malisa et al.
reedbuck, kongoni, oryx,warthog & hippopotamus (664e246 bp) (2006)
Chamois, pyrenean ibex, mouflon, ApoI, MseI/MaeII 12S rRNA (720 bp) D- Fajardo et al.
cattle, sheep and goat loop region (370 bp (2007)
Guinea fowl, quail, chicken, duck and turkey HinfI, Mph1103I, 12S rRNA (456 bp) Girish et al. (2007)
MvaI, Eco47I
Red deer, cattle, domestic pig, horse, chicken, MboI, Tsp509I 12S rRNA (455 bp) Park et al. (2007)
duck and turkey
Wild boar and domestic pig BspHI, BstUI MC1R (795 bp) Fajardo et al.
(2008a)
Spotted deer, hog deer, barking deer, sika deer, BsrI, BstSFI, DdeI, RsaI, 12S rRNA (440 bp) Gupta et al. (2008)
musk deer and sambar deer

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Red deer, sika deer, reindeer, elk and siberian NlaIV, TaqI Cytochrome b (466bp) Shin et al. (2008)
maral deer D-loop region(1175 bp)
Quail, pheasant, red- Legged partridge, chukar AluI, BfaI/HinfI, 12S rRNA (720 bp) Rojas et al.
partridge, guinea fowl, capercaillie, Eurasian Hpy188III, MboII D-loop region (310 (2008;2009a)
woodcock, woodpigeon,chicken, turkey muscovy bp)
duck
Red brocket deer, pygmy brocket deer and gray AflIII, BstnI, EcoRII, Cytochrome b (224 Gonza´lez et al.
brocket deer SspI bp) (2009)
Indian crocodile species (mugger, saltwater & HaeIII, MboI, MwoI Cytochrome b (628 bp) Mganathan et al.
gharial) (2009)
Buffalo, cattle, goat, domestic pig, quail, chicken AluI, BsofI, BstUI, Cytochrome b (359 Murugaiah et al.
and rabbit MseI, RsaI bp) (2009)
PCR-RFLP lab-on-a-chip technology
PCR-RFLP lab-on-a-chip technology is now a day readily used technology in which
standard chips can be utilized to find out the meat species. Agilent 2100 Bioanalyzer lab-on-a-
chip equipment can be used for this pupose. It is based on the principle of computer-generated
gel image using the 2100 Expert software including the 12S rRNA gene fingerprints generated
by the MseI restrictions. The readily available chips can detect the meat species having
molecular weight marker 50-1000 bp. Fajardo, et al. (2006) identified the meat species from
undigested samples of red deer, fallow deer, roe deer, chamois, mouflon, pyrenean ibex, goat ,
cattle, sheep and domestic pig. Dooley et al. (2004) used this technique for the authentication of
meat species like cattle, sheep, chicken, turkey or fish. Fajardo et al. (2006) is the only
published study to date describing the identification of game meats by means of this technique.

Species Identification by Randomly Amplified Polymorphic DNA (RAPD)

RAPD is a type of PCR reaction, but the segments of DNA that are amplified are
random. In this technique arbitrary primers are used to amplify DNA fragments in different
species and clear distinct patterns with high level of polymorphism can be detected between
species. The work done on this aspect by various researchers is tabulated in table 6.

Table 6 Meat species identification using PCR-RAPD

Workers Meat species speciation Technology adopted
Arslan et al. (2005), Koveza et al. Meat, fish and vegetable food PCR-RAPD using eight primers
(2005) and Mohindra et al., 2007 stuffs with sizes ranging from 19 to 26 bp
Chai et al. (1997) For ten bird species: pheasant, PCR-RAPD Fingerprint patterns
partridge, quail, guinea fowl,
pigeon, emu, ostrich, chicken,
local duck and mallard duck
Martı´nez and Yman (1998) Elk, kangaroo, reindeer, RAPD Species- specific profiles
buffalo and ostrich, as well where obtained in fresh, frozen and
as some domestic meat canned samples.
species
Martı´nez and Danielsdottir Seal and whale meat products By RAPD and PCR SSCP
(2000) (frozen, smoked, salted, dried, techniques using consensus
etc.) primers designed on the
mitochondrial cytochrome
gene.
Huang et al. (2003) Ostrich, quail, dove, emu and Using RAPD-PCR fingerprinting
pheasant
Arslan et al. (2005) Meats from wild boar, PCR-RAPD using a unique 10 bp
bear, camel and oligonucleotide.
domestic species

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Rastogi et al. (2007) Identify snake and buffalo, Targeting the mitochondrial
among other species 16S rDNA and NADH
dehydrogenase subunit 4 (ND4)
genes and the nuclear actin gene.

Species Identification by using Forensically Informative nucleotide sequencing (FINS)

FINS is a technique that combines DNA sequencing and phylogenetic analysis. In this
technique meat samples are identified on informative nucleotide sequences basis. Actually PCR
amplification and sequencing of conserved gene is one of the first techniques for meat species
identification. Among them mitochondrial DNA is highly conserved, gene on it Cytochrome-b
and 12S-r RNA used for meat species identification can be exploited for the meat species
speciation.

Real time PCR

Real time PCR is a improved version of PCR in which the reactions can be monitored at
early stages and reactions takes place can be monitored at every step. The early detection or
prediction of results can be achieved at early stage of the reactions. A rapid real-time polymerase
chain reaction (PCR) technique using SYBR Green detection system has been developed by
Fajardo et al. (2008b) for the quantification of red deer, fallow deer, and roe deer DNAs in
meat mixtures. The method combines the use of cervid-specific primers that amplify a 134,
169, and 120 bp of the 12S rRNA gene fragment of red deer, fallow deer and roe deer,
respectively, and universal primers that amplify a 140 bp fragment on the nuclear 18S rRNA
gene from eukaryotic DNA. There are several workers done their work on this aspect for
differentiation of meat ofwild and domestic animals. Some of the salient worked on
primers used and meat species identified is summarized in table 7 and 8.

Table 7 Common DNA sequences of the primers used in Real time PCR
Primers Length Sequence (50-30) Description Amplicon Amplicon
(bp) size (bp) Tm (C)
12SCEQFW 32 CAAAAACATATAACG Red deer specific 134 76.5-78
AAAGTAACTTTCCGA CC forward primer
12SCEQREV 28 AGTACTCTGGCGAAT Red deer specific
AGTTTTGTCTGCA reverse primer
12SDDQFW 24 TAAACAACGAAGGTA Fallow deer specific 169 78–79.5
ACCTTATCG forward primer
12SDDQREV 19 AAAGCACCGCCAAG Fallow deer specific
TCCTT reverse primer
12SCCQFW 23 GCGTAAAGCGTGTTA Roe deer specific 120 72–73
AAGCATAC forward primer
12SCCQREV 25 GCTATCGTGTTTCAG Roe deer specific
CTATTTTCAA reverse primer
18SEUDIR 23 TCTGCCCTATCAACT Eukaryotes forward 140 84–83
TTCGATGG primer
18SEUINV 18 TAATTTGCGCGCCTG CTG Eukaryotes reverse
primer

Taq Man assays

TaqMan assays for meat species identification was developed by Dooley et al. (2004) for
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detection of beef, pork, lamb, chicken and turkey. They developed the assays around small
(amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. In this
technique speciation was achieved using species-specific primers. For meat species speciation
they developed two Taq Man probes; the first was specific to the mammalian species (beef, lamb
and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan
PCR conditions were applied in this assays and PCR was limited to 30 cycles. On application of
assays to DNA extracts from raw meat admixtures, it was possible to detect each species when
spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was
not determined; however, experimentally determined limits for beef, lamb and turkey were
below 0.1% (Kesmen et al., 2009). The work carried out by Ali et al. (2012) on Taq Man assay
for meat species speciation is depicted in table 9.

Table 8 Meat species identifications using real time PCR

Workers Meat species speciation Technology adopted
Jonker et al. (2008); Beef, pork, lamb, horse, Real time PCR assay
Laube etal., (2007) chicken, turkey and duck

Wetton et al. (2002) Tiger DNA from tiger using a species-

specific oligonucleotide pair targeting the
mitochondrial cytochrome b gene and the
SYBR Green fluorescent intercalator
Hird et al. (2004) Deer and some domestic species Real-time TaqMan technology with
truncated primers located on mitochondrial
cytochrome b gene
Lo´pez-Andreo et al. Ostrich and other meat species TaqMan realtime PCR systems on the
(2006) mitochondrial cytochrome b gene
Lo´pez-Andreo et al. Kangaroo, horse, bovine and Using mitochondrial cytochrome b
(2006) porcine species in mixed sam sequences and the SYBR Green
fluorescent molecule
Chisholm et al. (2008) Pheasant and quail Using species-specific primers and
TaqMan probes designed on the
mitochondrial cytochrome b gene
Fajardo et al. (2008b, Red deer, fallow deer, roe SYBR Green real-time PCR assay
2008c) deer, chamois and pyrenean using species-specific primers targeting
ibex in meat mixture the mitochondrial 12S rRNA and D-loop
gene
Rojas, et al. (2010b) Quail, pheasant, partridge, The assay is based on specific primers
guinea fowl, pigeon, Eurasian and probes designed for each target
woodcock and song thrush species on the mitochondrial 12S rRNA
gene

Table 9 Primers and probes for cytochrome b (cytb) single species assays in the Taq Man assay conducted
by Ali et al. (2012)
Species Optimal Reporter Sequence (5’–3’) moiety Tm Optimal Amplicon
primer sets concentration size (bp)
(nM)
Primer Probe
Beef Forward CGG AGT AATCCT 59.8 300 116
Reverse TCT GCTCACAGT
GGA TTGCTG ATA AGA GGT TGG TG 58.6 900
Lamb Forward GAG TAA TCCTCC 56.3 300 175 133
Reverse
TAT TTT GCG ACA AGG TTT GTGCCA ATA TAT GGA ATT 56.7 300

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Pork Forward 2 ATG AAA CAT TGG AGT AGT CCT ACT ATT TAC C 58.9 300 175 149
Reverse 2 CTA CGA GGT CTG
58.4 900
TTC CGA TAT AAG G
Chicken Forward 1 AGC AAT TCC CTA CAT TGG ACA CA 59.4 300 200 133
Reverse 3 GAT GAT AGT AAT ACC TGC GAT TGC A 58.3 300
Turkey Forward ACC CTA GTA GAG TGA GCC TGA GG AAG GGC AGG 56.9 300 150 86
Reverse AGG AAG TGG AG 59.3 300
Mammal Probe TGA GGA CAA ATA TCA TCA TTC TGA GGA GCW ARG >68
FAM TYA
Poultry Probe ACA ACC CAA CCC TTA CCC GAT TCT TC 65.8
TET
Beef Forward CGG AGT AAT CCT TCT GCT CAC AGT GGA TTG 59.8
Reverse CTG ATA AGA GGT TGG TG 58.6
FAM, 6-carboxyfluorescein; TET, 6-carboxy-4,7,20,70-tetrachlorofluorescein; Tm, melting temperature; bp, base-pairs.

Conclusion
The meat species speciation is not an easy task. The use of an appropriate
technology for a particular type of meat species detection is cumbersome and needs thorough
knowledge of thE structure and composition of the muscle tissues and its molecular structure.
The applicability of the technologies is dependent on the type of sample available and
requirement of the tests to be done. However, for simple samples easy and reproducible
methods are adopted and if samples are cooked and deteriorated then complicated molecular
techniques are applied. So the decision of techniques to be applied must base on feasibility of
the tests and authentications.

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