MEAT

SCIENCE
Meat Science 70 (2005) 107–112
www.elsevier.com/locate/meatsci

Meat species identification by polymerase chain reaction-restriction

fragment length polymorphism (PCR-RFLP) of mitochondrial
12S rRNA gene
a,*
P.S. Girish , A.S.R. Anjaneyulu a, K.N. Viswas b, B.M. Shivakumar c, M. Anand d,
M. Patel e, B. Sharma d
a
Division of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
b
National Biotechnology Center, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
c
Division of Animal Genetics, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
d
Division of Biochemistry and Food Sciences, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India
e
Division of Livestock Production and Management, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India

Received 5 May 2004; received in revised form 12 December 2004; accepted 12 December 2004

Abstract

Adulteration of high quality meat and meat products with their inferior/cheaper counterparts is a problem in the meat industry.
The present study investigated the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the
mitochondrial 12S rRNA gene for identification of the origin of meats. PCR-RFLP was applied for species identification of beef,
buffalo meat, mutton and chevon. PCR amplification yielded a 456-bp fragment in each of these species. The amplicons were
digested with AluI, HhaI, ApoI and BspTI restriction enzymes resulting in a pattern that could identify and differentiate each of
the above species. This technique did not yield satisfactory results with meat mixtures/meats. However, consistent results were
obtained with both fresh and processed meat samples.

2005 Elsevier Ltd. All rights reserved.

Keywords: Meat species; Mitochondria; 12S rRNA; PCR-RFLP

1. Introduction nique in many fields of molecular biology owing to its

Molecular techniques developed over the last two
decades have allowed the development of authentic
and reliable methods for meat species identification.
These techniques are promising and can overcome the
drawbacks of many conventional methods. Polymerase
chain reaction (PCR) is the most commonly used tech-
*
Corresponding author. Present address: Livestock Products Tech-
nology Department, G.B. Pant University of Agriculture and Tech-
nology, Pantnagar, Udham Singh Nagar (Dt), Uttaranchal, India.
Tel.: +91 9412959120; fax: +91 5812447284.
E-mail address: girishlpt@yahoo.com (P.S. Girish).
sensitivity, specificity and capability to detect even a sin-
gle copy of DNA sequence from a single cell sample
(Chikuni, Tabata, Kosugiyama, & Monma, 1994).
Some of the molecular approaches applied in the
past for meat species identification include PCR (Co-
lombo, Vincava, sacchelli, Colombo, & Camisasca,
1998; Gouli, Mengguang, Zhiang, Hongsheng, &
Qiang, 1999), polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) (Chung,
Kim, Kim, & Han, 2000; Hopwood, Fairbrother,
Lockley, & Bardsley, 1999), random amplified poly-
morphic DNA-PCR (RAPD-PCR) (Ganai, Singh, &
Butchaiah, 2000; Koh, Lim, Chua, Chew, & Phang,

0309-1740/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2004.12.004
108 P.S. Girish et al. / Meat Science 70 (2005) 107–112
1998), DNA hybridization (Chikuni, Ozutsume, Hois-
hiKawa, & Kato, 1990; Matsunga et al., 1998) and
nucleotide sequencing (Colombo, Marchisio, Pizzini,
& Cantoni, 2002; Girish et al., 2004).
In PCR-RFLP, a conserved region of DNA sequence
is amplified using PCR, followed by digestion with
restriction enzymes, which can reveal genetic variation
between species (Partis et al., 2000).
Both nuclear and mitochondrial genes have been tar-
geted for species detection by PCR-RFLP. Among the
mitochondrial genes, the cytochrome b gene (Meyer,
Hoefelein, Luethy, & Candrian, 1995; Branciari, Avel-
lini, Sukasi, Antonio, & Rea, 2000), D-loop (Murray,
McClymont, & Strobeck, 1995), 12S rRNA gene (Ab-
dulmawjood & Buelte, 2001) and 16S rRNA gene (Bor-
go, Souty Grosset, Bouchon, & Gomot, 1996) have been
used for species identification.
In this study, we report a method for species identifi-
cation of fresh/processed beef, buffalo meat, mutton and
chevon samples by PCR-RFLP of mitochondrial 12S
rRNA gene. Methodology and advantages of PCR-
RFLP are also discussed.
2. Materials and methods
2.1. Materials
Most chemicals were purchased from Sigma or
Merck. Proteinase K, Ribonuclease A, Taq DNA poly-
merase, dNTPs and DNA markers were procured from
Bangalore Genei (Bangalore, India). Primers were syn-
thesized by Operon Inc. (Singapore). Restriction en-
zymes AluI, BspTI and ApoI were purchased from
MBI Fermentas (Canada), whereas, HhaI was obtained
from Bangalore Genei (Bangalore, India).
2.2. DNA isolation from meat samples
DNA isolation from meat samples was carried out as
described by Chikuni et al. (1994). Briefly, the meat sam-
ples were triturated and mixed with lysis buffer contain-
ing 10 vol of 10 mM Tris–HCl, (pH 8.0), 100 mM
ethylene diamine tetra acetate (EDTA), 0.5% sodium
dodecyl sulphate (SDS) and 0.1 mg/ml of Proteinase K
for 3 h at 50 C. The resultant mixture was treated with
50 lg/ml of RNase A for 1 h at 37 C, which was then
extracted with an equal volume of phenol:chloro-
form:isoamyl alcohol (25:24:1 vol/vol) and again with
an equal volume of chloroform. DNA precipitated by
ethanol and 1 M ammonium acetate solution, was dis-
solved in TE buffer (10 mM Tris–HCl, pH 7.4 and
0.1 mM EDTA). DNA concentration was determined
by measuring the absorbance at 260 nm.
DNA from processed meats was extracted in buffer
containing 100 mM Tris–HCl, pH 9.0, 100 mM sodium
chloride, 5 mM EDTA, 1% SDS and 10 mM DTT
(dithiothreitol), followed by purification with phe-
nol:chloroform:isoamyl alcohol extraction and ethanol
precipitation.
2.3. Polymerase chain reaction
Universal primers for mt 12S rRNA gene (Forward-
5 0 -CAA ACT GGG ATT AGA TAC CCC ACT AT-3 0 ,
Reverse-5 0 -GAG GGT GAC GGG CGG TGT GT-3 0 ),
were used for PCR amplification, as described by Ko-
cher et al. (1989). Amplification was carried out in
0.2 ml PCR tubes containing 5 ll of 10· PCR buffer
(100 mM Tris–HCl, pH 9.0, 15 mM MgCl2, 500 mM
KCl and 0.1% gelatin), 1 ll of 10 mM dNTP mix, 1 ll
(20 pmol) each of forward and reverse primers, 1 U of
Taq DNA polymerase, 50 ng of purified DNA and auto-
claved Milli-Q water to make a volume up to 50 ll. Con-
ditions on a Master Cycler gradient thermocycler
(Eppendorf, Germany) were as follows: 5 min at 94 C
for initial denaturation, followed by 30 cycles of ampli-
fication (45 s at 94 C, 45 s at 60 C and 1 min at 72 C)
and final extension for 10 min at 72 C. The PCR prod-
ucts were analyzed by electrophoresis in 1% agarose gel
with ethidium bromide staining.
2.4. Sequencing and restriction analysis
The amplicons of mt 12S rRNA gene were sequenced
using ABI prism 377 automated DNA sequencer at the
Sequencing facility, University of Delhi, New Delhi, In-
dia. The sequences were analyzed using the Editseq pro-
gram and restriction mapped using the Mapdraw
program of Lasergene software (DNAstar, Inc., New-
York). Restriction enzymes with unique restriction pat-
terns with respect to each species were selected using
tabulation and comparison. The sequences were submit-
ted to European molecular biology laboratory (EMBL)
database and were assigned with accession numbers,
viz., AJ490501 (Cattle), AJ490502 (Buffalo), AJ490503
(Sheep) and AJ490504 (Goat).
2.5. Restriction fragment length polymorphism
PCR amplicons of the mitochondrial 12S rRNA gene
were subjected to restriction enzyme digestion with suit-
able restriction enzymes according to the suppliersÕ
instructions. Briefly, enzyme-buffer mix was prepared
by mixing 2 ll of restriction enzyme with 8 ll of the
respective buffer.
Reaction mix was prepared by mixing 10 ll PCR
product with 2 ll of enzyme buffer mix. Volume was
made up to 20 ll with autoclaved MilliQ water and incu-
bated overnight at 37 C. Digested product was visual-
ized by electrophoresis in 2% agarose gel along with
100 bp ladder (Bangalore Genei).
 P.S. Girish et al. / Meat Science 70 (2005) 107–112 109

3. Results chosen for PCR-RFLP studies, so as to detect and dif-

Universal primers used in this study amplified a 456-
bp fragment of mitochondrial 12S rRNA gene in all four
species, viz., cattle, buffalo, sheep and goat (Fig. 1). PCR
amplification was observed at various annealing temper-
atures from 50 to 62 C. However, 60 C was chosen for
further experiments. At least 10 samples from each spe-
cies were subjected to PCR amplification and used for
subsequent RFLP studies. The uniform amplification
was observed in meats processed at 72, 90, 120 C for
30 min and meats subjected to deep fat frying. However,
the intensity of the signal reduced with increased cook-
ing temperature and was lowest in meat samples sub-
jected to 120 C for 30 min.
Restriction map of sequenced amplicons of cattle,
buffalo, sheep and goat mitochondrial 12S rRNA partial
sequences along with selected restriction sites are given
in Table 1. Based on analysis of the restriction map of
sequences AluI, HhaI, ApoI and BspTI enzymes were
ferentiate meat species (Table 1).
AluI enzyme generated fragments of 359 and 97 bp in
cattle, 246 and 210 bp fragments in both sheep and goat
and no fragments in buffalo, as per expectation. HhaI,
ApoI and BspTI were specific only to buffalo, sheep
and goat generating 247 and 209 bp, 329 and 127 bp
and 323 and 133 bp fragments, respectively (Fig. 2).
The results were representative of 10 separate experi-
ments with different samples.
Different cooking methods did not affect the RFLP
pattern and results were similar in meat samples pro-
cessed and cooked at 72 C (patties), 90 C (steam
cooked blocks), 120 C for 30 min (autoclaved blocks)
and of fried meat products (fries/croquettes) (Fig. 3).
Specificity did not vary with cooking and the presence
of other ingredients.
Similar studies were conducted on mixed meats. For
this purpose, various combinations of meat mixtures
were prepared. Buffalo meat with chevon/mutton, beef
with chevon/mutton, beef with buffalo meat and mutton
with chevon combinations were prepared in 50:50,

Fig. 1. Polymerase chain reaction (PCR)-amplification of mitochon-

drial 12S rRNA gene. Amplicons were analyzed by 1% agarose gel
electrophoresis. M: 100 bp ladder; 1: Beef; 2: Buffalo meat; 3: Mutton;
4: Chevon.

Table 1
Restriction pattern of mitochondrial 12S rRNA gene for the different
species Fig. 2. Restriction fragment length polymorphism (RFLP) of mito-
Species enzymes Cattle Buffalo Sheep Goat chondrial 12S rRNA gene. PCR amplicons were subjected to restric-
tion analysis with AluI in beef resulting in 359 and 97 bp fragments
AluI 359 + 97 – 246 + 210 246 + 210
(Lane 1), HhaI in buffalo meat resulting in 246 and 210 bp fragments
HhaI – 247 + 209 – –
(Lane 2), ApoI in mutton yielding 329 and 127 bp fragments (Lane 3)
BspTI – – – 323 + 133
and BspTI in chevon resulting in 323 and 133 bp fragments. 100 bp
ApoI – – 329 + 127 –
DNA ladder is shown in lane M.
110 P.S. Girish et al. / Meat Science 70 (2005) 107–112

Fig. 3. Restriction fragment length polymorphism of mitochondrial 12S rRNA gene of processed meat products. DNA isolated from patties (Lane
1), steam cooked blocks (Lane 2), Croquettes (Lane 3) and autoclaved blocks (Lane 4) was amplified with universal primers for mitochondrial 12S
rRNA gene and subjected to restriction analysis with AluI for beef yielding 359 and 97 bp fragments, HhaI for buffalo meat (a) resulting in 246 and
210 bp fragments, ApoI for mutton (b) yielding 329 and 127 bp fragments and BspTI for chevon (c) resulting in 323 and 133 bp fragments. 100 bp
DNA ladder is shown in lane M.

10:90, 5:95 and 1:99 proportions. Results were not con-
sistent because even after thorough mincing and mixing
of different meats in the predetermined proportion, the
amount taken for extraction of DNA may not be repre-
sentative of the mix. Also during the PCR, the amplifi-
cation may not be proportional to the respective
quantity of DNA present. Hence the obtained results
were ambiguous and did not correspond to the propor-
tions of the mixtures (data not shown).
However, efforts are underway to standardize the
technique for detection of meat species in adulterated
samples.
4. Discussion
Mitochondrial DNA sequence is highly conserved in
different species of animals (Antoinette, Van der kuyl
Carla, Kuiken, & Dekker, 1995). This has enabled
designing of universal primers for the 12S rRNA gene,
which can amplify corresponding fragments in a wide
variety of organisms including birds and insects (Kocher
et al., 1989). General differences between mitochondrial
12S rRNA gene sequences is sufficient for species identi-
fication of different biological samples (Prakash et al.,
2000). Molecular techniques, such as polymerase chain
reaction (Rodriguez et al., 1991), randomly amplified
polymorphic DNA (RAPD) fingerprinting (Ganai
et al., 2000), DNA hybridization (Ebbehoj & Thomson,
1991) and gene sequencing (Chikuni et al., 1994) have
been tried elsewhere for meat identification. Each of
these methods has their own limitations. PCR-RFLP
of the mitochondrial 12S rRNA gene is highly repeat-
able, cheaper and quicker than the methods cited above
(Meyer et al., 1995). Although, DNA sequencing and
analysis is accurate and authentic, it is costly, time con-
suming and not suitable for routine species identification
studies. PCR-RFLP has been proven to be a practical,
simple and rapid technique (Meyer et al., 1995; Partis
et al., 2000).
In the present study, a partial sequence of mitochon-
drial 12S rRNA gene was targeted for the PCR-RFLP
study, so as to identify the meat species.
Primers used in this study amplified cattle, buffalo,
sheep and goat mitochondrial 12S rRNA gene frag-
ments perfectly. Using universal primers for PCR ampli-
fication obviated the requirement for an internal
control, which is otherwise used to monitor the success
of DNA amplification. As each cell contains about
one thousand copies of mitochondrial DNA, PCR as-
says based on its amplification were shown to be more
sensitive as compared to single or low copy nuclear
DNA targets (Partis et al., 2000). Since, the quantity
of PCR products generated corresponds to the copy
number of the target DNA sequence (Partis et al.,
2000), a higher copy number of mitochondrial DNA en-
sures a sufficiently high quantity of PCR product, even
when small amounts of fresh/or processed meat samples
are used.
RFLP pattern of restriction enzymes used in this
study was highly specific and specificity did not vary
with the processing temperatures of the meat samples.
 P.S. Girish et al. / Meat Science 70 (2005) 107–112
Because of the high copy number of small, circular mito-
chondrial DNA in cells, the chances of their survival un-
der different processing conditions are higher, making it
ideal for processed meat species identification. Wolf,
Rentsch, and Huebner (1999) successfully identified
roasted game meats by PCR-RFLP of the 464 bp frag-
ment of the mitochondrial cytochrome b gene.
Sequence analysis of the mitochondrial 12S rRNA
gene showed a significant variation between different
species of animals, enabling application of PCR-RFLP
to distinguish them. AluI, HhaI, ApoI and BspTI were
chosen as they gave higher distinct fragment sizes, en-
abling easy interpretation of results after restriction
digestion. In a similar report, Meyer et al. (1995) ampli-
fied the 359-bp fragment of the cytochrome b gene using
PCR followed by restriction digestion with AluI, RsaI,
TaqI and HinfI to identify pig, cattle, wild boar, buffalo,
sheep, goat, horse, chicken and turkey meat. In another
report, Partis et al. (2000) generated DNA fingerprints
for 22 animal species by amplifying 359 bp regions
within the cytochrome b gene and digesting the ampli-
fied product with HaeIII and HinfI. All species could
be discriminated except kangaroo and buffalo.
PCR-RFLP of 12S rRNA gene could differentiate
closely related meat species, such as cattle–buffalo and
sheep–goat. As shown in Table 1 restriction enzymes
viz., HhaI, ApoI and BspTI have unique sites in buffalo,
sheep and goat sequences, respectively. AluI gives 97
and 359 bp fragments in cattle and 246 and 210 bp frag-
ments in sheep and goat. Hence, interpretation of results
was unambiguous and simple. In a similar experiment,
PCR-RFLP of mitochondrial 12S and 16S rRNA gene
were used for differentiation of fresh, cooked and
canned meat products prepared from closely related
snail meat species, Helix pomatia and Helix luconum
(Abdulmawjood & Buelte, 2001; Borgo et al., 1996).
Chikuni et al. (1994) differentiated sheep and goat meat
by PCR-RFLP of satellite I DNA sequence using ApaI
restriction enzyme. Restriction profile of melanocortin
gene was used as DNA marker for differentiation of
Hanwoo meat from Holstein and Angus meats (Chung
et al., 2000).
Applicability of this method in mixed/adulterated
meats was found not to be satisfactory. Results were
not representative of the actual amount of the different
components present in the mixture. This could be due
to the disparity in the quantity of PCR product ampli-
fied and the amounts of target DNA present (Partis
et al., 2000). Also, this method could not differentiate
male and female tissues. This may be due to maternal
inheritance of mitochondrial DNA and hence, similarity
in sequences of male and female mitochondrial DNA.
It can be concluded that, these four commercial spe-
cies, i.e., cattle, buffalo, sheep and goat can be qualita-
tively identified and differentiated by PCR-RFLP of
mitochondrial 12S rRNA gene. This method can be ap-
111
plied with equal efficiency to both fresh and processed
meats, however, with little success to meat mixtures.
Acknowledgments
We thank the Director, Indian Veterinary Research
Institute for providing necessary facilities and ICAR
for financial assistance in the form of senior research fel-
lowship to Girish P.S. We also thank Drs. Kondaiah N.,
Bhilegaonkar, K.N., Satish Kumar, Mallik S.V.S. and
Aggarwal, R.K. for helpful discussions.
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