MEAT

SCIENCE
Meat Science 76 (2007) 666674
www.elsevier.com/locate/meatsci

Species identication and authentication of tissues of animal

origin using mitochondrial and nuclear markers
Gurdeep Rastogi 1, Mahesh S. Dharne 1, Sandeep Walujkar, Ashutosh Kumar,
Milind S. Patole, Yogesh S. Shouche *
Molecular Biology Unit, National Centre for Cell Science, Ganeshkhind, Pune 411 007, Maharashtra, India
Received 20 March 2006; received in revised form 24 January 2007; accepted 9 February 2007

Abstract
We evaluated and compared the utility of mitochondrial markers viz. 16S rDNA and NADH dehydrogenase subunit 4 (ND4) and a
nuclear marker viz. the actin gene to identify the specimens of animal origin for forensic identication, food regulatory control and to
prevent illegal trading, poaching and conservation of endangered species. We also tested PCR ngerprinting methods like RAPD and
actin barcoding to generate species-specic ngerprints. Our results suggested that mitochondrial markers are more ecient than
nuclear markers for the purpose of species identication and authentication. Among PCR ngerprinting approaches, RAPD was proved
to be more discriminatory, accurate and ecient than actin ngerprinting. Considering the present scenario in trading of vertebrate animal tissues like bualo, cow, pig, goat, chicken, frogs, shes and snakes etc., mitogenomics based technology proved to be ecient and
reliable in resolving problems like meat adulteration and smuggling across countries.
2007 Elsevier Ltd. All rights reserved.
Keywords: Mitochondrial markers; Nuclear marker; 16S rDNA; ND4; RAPD; Meat

1. Introduction
Reliable labeling of meat products oered for sale is
important in order to assure the consumers about the identity and quality of the food products they consume for a
variety of reasons including adulteration and socio-religious
factors. Due to an increase in the consumption demand and
high cost, other cheaper animals are being used as fraudulent substitutes or mislabeled. When an endangered animal
has been killed for food or sport, phenotypic markers are
often destroyed or intentionally removed to create problems
for personnel who are involved in determining the species
origin of an animal or products derived from it in order to
enforce conservation and or health-related regulations
(Palumbi & Cipriano, 1998). Curbing the illegal trading of
ecologically and economically important animals is neces*

Corresponding author. Tel.: +91 20 25690922.

E-mail address: yogesh@nccs.res.in (Y.S. Shouche).
These authors have contributed equally to this work.

0309-1740/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2007.02.006

sary from the point of view of biological conservation and

also for food industries. Slow neurological disorders like
bovine spongiform encephalopathy could be transmitted
through contaminated food intake by animals; therefore,
the examination of meat components in commercial feeds
is important for the rapid control of outbreaks of disease
(Wong, Wang, But, & Shaw, 2004).
The conventionally available methods for species identication and authentication include various forms of electrophoresis and use of immune sera in agar gel diusion
(Patterson, 1995). However, these methods are based on
protein expression pattern and suer from the disadvantage that after cooking the proteins are heat denatured.
This results in the alteration of electrophoresis pattern.
Moreover, immune sera often show cross species reactivity
(Charnyi, 1967). Species identication and authentication
based on DNA analysis is more sensitive and reliable since
it is independent of the tissues being compared. DNA
remains intact even at high temperatures, therefore
allowing the analysis of processed and heat-treated food

G. Rastogi et al. / Meat Science 76 (2007) 666674

products. One of the stages of dealing with biological materials submitted to forensic laboratories is species identication of the biological evidence e.g. blood strains on a cloth
collected from a fatal road accident and insurance claims
that involve road accidents with animals require correct
species identication. Mitochondrial DNA (mt-DNA)
forensic analysis can be used in such disputes even without
the reference sample. The characteristic high copy number,
maternal inheritance and high degree of sequence variability make mt-DNA a powerful tool for forensic identication. Most of these methods make use of PCR
amplication and direct sequencing of a conserved gene
from the sample e.g. mt-12S rDNA (Rastogi et al., 2004),
mt-16S rDNA (Borgo, Souty Grosset, Bouchon, & Gomot,
1996) cytochrome b gene (cytb) (Wong et al., 2004), use of
oligonucleotide probes (Hunt, Parkes, & Lumley, 1997),
amplication of species-specic DNA sequences (Arevalo,
Davis, & Sites, 1994; Meyer, Hofelein, Luthy, & Candrian,
1995), microsatellites analysis (Lorenzini, 2005), RFLP
analysis (Meyer et al., 1995), SSCP of PCR amplied fragments (Cespedes et al., 1999) and RAPD (Calvo, Zaragoza,
& Osta, 2001; Franklin, Taylor, & Mills, 1999). The denition of mono-locus-specic primers is quite reliable but the
technique is conditioned by a prior knowledge of nucleotide sequences anking the loci and richness of gene databases. The advantage of RAPD ngerprinting lies in the
fact that it is a sequence independent approach and each
primer DNA annealing will produce a dierent spectrum
of fragments from the PCR generating a species-specic
ngerprint. RAPD is faster and cheaper than the other
DNA based methods, but major the drawback is the poor
reproducibility of the results obtained (Louie et al., 1996;
Welsh & McClelland, 1990). The 16S rDNA and ND4
genes have been very well studied among dierent vertebrate groups at the population level (Goebel, Donnelly,
& Atz, 1999; Hillis, Mable, & Moritz, 1996; Simon et al.,
1994). These studies have indicated that the level of 16S
rDNA and ND4 gene sequence variation is suitable for
addressing general questions on interspecic diversity. To
date, no studies have been reported using the ND4 gene
sequences to identify the species origin of animal tissues.
To the best of our knowledge, this is the rst report where
ND4 gene sequences were used for the purpose of species
identication of animal tissues.
Reliance on a single, maternally inherited character (mtDNA) can provide misleading results in situations where
there is a possibility of hybridization or introgression,
and analyses of both nuclear and mitochondrial markers
are therefore desirable (Palumbi & Cipriano, 1998). Several
nuclear genes, which are conserved throughout the vertebrates and invertebrates like actin and histones, have been
reported in investigations involving adulteration and conservation issues (Fairbrother, Hopwood, Lockley, &
Bardsley, 1998; Svenson & Whiting, 2004). Genome
sequencing projects on many vertebrates revealed high
sequence homology in protein coding regions of actin genes
but considerable variability in intron number and sizes.

667

PCR amplication of the intron which separates exons 6

and 7 generates characteristic species-specic ngerprints that are highly stable and reproducible (Fairbrother et al., 1998; Svenson & Whiting, 2004).
Previously, we have shown the application of mt-12S
rDNA and conformation sensitive gel electrophoresis
(CSGE) for species authentication and adulteration of
raw and cooked meat tissues (Rastogi et al., 2004). The
present study deals with the evaluation of mitochondrial
markers (16S rDNA and ND4) and a nuclear marker
(actin) for the sensitive and accurate determination of species origin of commercial meat samples like bualo,
chicken, cow, goat and pig. Forensic specimens like blood
and viscera of unknown species origin were also included in
order to ensure the utility of the technique in forensic analysis. The study also aims at species authentication of animals like frogs, shes and snakes that are consumed as
rich and energetic food source in many parts of the world,
that are being traded, smuggled and sold in markets all
over the world. The consumption of meat of domestic animals like bualo, cow, pig and chicken may have ethical
issues related to dierent religious beliefs and their adulteration might lead to severe ethical problems (Aida, Che
Man, Wong, Raha, & Son, 2005). The utility of PCR ngerprinting methods like RAPD and actin barcoding to
generate ngerprints characteristics of dierent meat
species was tested and their potential application as reliable, fast and simple methods for the determination of origin of meat species was also discussed.
2. Materials and methods
2.1. Sample collection
Meat tissue specimens from slaughtered animals of different origins like bualo (Bubalus bubalis), cow (Bos indicus), pig (Sus scrofa), goat (Capra hircus) and chicken
(Gallus gallus) were collected from commercial wet markets
in Pune, India by food inspections ocers. Samples were
collected from authorized government meat shops during
regular survey programmes. Forensic samples like blood,
brain and viscera were also collected from the regional
forensic laboratory of Pune. The molecular biology unit
of NCCS being a partner in the study was used as a blind
control to nd out the accuracy of the protocols, results
and outcome of the study. Identity of samples was revealed
only when NCCS accomplished the identication of these
specimens. Thus, all the samples included served both as
unknown and reference samples in our study. A dead frog
specimen belonging to genus Bufo melanostictus was collected from Pune University garden, India. A sh specimen
belonging to Mystus cavasius was purchased from local sh
market in Pune, India. Moult sample of snake belonging to
genus Uropeltis was collected from forest areas of Pune
city. Specimens like frog, sh and snake moult were morphologically identied by their phenotypic characteristics
with the help of an animal taxonomist. Tissue samples

668

G. Rastogi et al. / Meat Science 76 (2007) 666674

immediately after arrival at the laboratory were stored in

80 C until further use.
2.2. DNA extraction, PCR amplication, sequencing and
ngerprinting
Extraction of total DNA was performed from 25 mg of
each tissue samples. Briey, all the specimens were subjected
to enzymatic digestion by addition of 500 ll of lysis buer
(150 mM NaCl, 50 mM TrisHCl, 10 mM EDTA (pH
8.0), 50 ll of 20% (w/v) SDS and 15 ll of Proteinase K
(20 mg/ml stock)) and incubated at 55 C for 8 h with gentle
inversion. The above digested materials were processed for
DNA isolation using standard phenolchloroform extraction method (Sambrook, Fritsch, & Maniatis, 1989). The
extracted DNA was dissolved into 50 ll of TrisEDTA
(pH 8.0) buer. About 100 ng of DNA was used as template
for the PCR amplication of mitochondrial markers (16S
rDNA and ND4) and the nuclear marker (actinintron
region). Amplication conditions and primers used were
based on previous studies and are summarized in Table 1.
PCR was carried out in 50 ll reaction volume containing
20 pM of each forward and reverse primer, 0.4 mM (each)
dNTPs (Invitrogen), 0.5 U of DyNazyme II DNA Polymerase (Finland) as per manufacturers protocol. PCR products
were checked by electrophoresis on 1% agarose gel in 1X
TrisBorateEDTA (TBE) buer and visualized under
UV light. In all PCRs, samples were amplied in duplicate

to check the consistency of results. All PCRs were performed at least on two dierent occasions in order to check
the reproducibility of the experimental procedure and the
ngerprint patterns. A negative control was also included
in all PCRs. PCR products were then puried for sequencing by the polyethylene glycolNaCl precipitation method
as described earlier (Rastogi et al., 2004). Puried PCR
products (100 ng) were directly sequenced using a 3730
DNA Analyzer (Applied Biosystems, Foster City, CA) as
per the manufacturers protocol. For RAPD, only single
primer was added in the PCR reaction. We have tested ve
dierent RAPD primers (Operon Technologies) for their
ability to provide readily interpretable and reproducible
RAPD proles. Only one primer was found to give a clear
and distinct RAPD ngerprint, and was used in further
investigations. In order to generate species-specic actin ngerprints, we amplied introns of the actin gene, which separate exon 6 and 7. Simultaneously, we made slight
modications to the PCR conditions (Table 1) in order to
get amplication of a single intron generating a single band
of 400 bp, which can be sequenced directly.
2.3. Dendrogram construction
PCR ngerprints were interpreted after normalization
and dendrograms were constructed for each ngerprinting
method using the UPGMA algorithm for cluster analysis
by Quantity One-4.4.0 gel documenting software (Bio-

Table 1
PCR primers and amplication conditions

Gene targeted

Primer sequence (5 0 3 0 )

mt-16S rDNA

16SAT
CGCCTGTTTATCAAAAACA
16SBT
CCGGTCTGAACTCAGATCACGT

mt-ND4

ND4
CATTACTTTTACTTGGATTTGCACCA
LEU
CACCTATGACTACCAAAA
GCTCATGTAGAAGC

nu-Actin (for direct

sequencing)

ActinF
CCTACAACAGCATCATGAAGTG

ActinR
GCTGATCCACATCTGCTGGAAG
Actin (for barcoding)

ActinF
CCTACAACAGCATCATGAAGTG

PCR conditions
9
94  C; 1 min =
55  C; 1 min 35X
;
72  C; 1 min
9
94  C; 1 min =

55 C; 1 min 35X
;
72  C; 1 min

Amplicon size (bp)

Reference

535

Goebel et al. (1999)

700

Arevalo et al. (1994)

400

Fairbrother et al. (1998)

9
95  C; 2 min =
45  C; 2 min
25X
;
68  C; 10 min

95705

Fairbrother et al. (1998)

9
95  C; 1 min =

36 C; 1 min 40X
;
72  C; 2 min

1761250

Franklin et al. (1999)

9
95  C; 0:30 min =

50 C; 0:45 min 25X
;
72  C; 0:30 min

ActinR
GCTGATCCACATCTGCTGGAAG

RAPD

F4
GGTGATCAGG

X indicates number of cycles in PCR. All PCRs included an initial denaturation at 94 C for 3 min and nal extension at 72 C for 10 min

G. Rastogi et al. / Meat Science 76 (2007) 666674

Rad, Hercules, CA). Computed similarities among ngerprints were estimated by means of the dice similarity coefcient. To survey the phylogenetic utilities of the dierent
markers, we used nucleotide sequence data for further phylogenetic analyses. The electropherograms obtained for
both the strands were checked by BioEdit (Brown, 1999)
and also by manual proof reading for any ambiguity or
inframe errors. Sequences were aligned using the Clustal
W program (Thompson, Higgins, & Gibson, 1994). Similarity searches for obtained partial sequences were carried
out using BLAST at NCBI (Altschul, Gish, Miller, Myers,
& Lipman, 1990). Ambiguously aligned portions were
deleted from the beginning and end using DAMBE (Xia,
2000). Phylogenetic trees were constructed by UPGMA
method using Molecular evolutionary genetic analysis
(MEGA) v3.1 (Kumar, Tamura, & Nei, 2004). Bootstrap
re-sampling analysis for 1000 replicates was performed to
estimate the condence of tree topologies. Gaps/missing
nucleotide positions in alignment were completely deleted.
Similarity tables showing percentage relatedness among
gene sequences were drawn for each marker using the
DNADIST program of PHYLIP (Felsenstein, 1989).
3. Results and discussion
Surveillance to detect adventitious or fraudulent contamination in meat products serves to reassure all parties
within the food supply chain. This is particularly true of
ethnic groups whose religious beliefs forbid the consumption of particular species and who require assurance that
they are purchasing food that conforms to their beliefs.
The ability to intercept adulterated meat before it enters
domestic commerce is a high priority issue. Short DNA
sequences from a dened stretch of the genome provide a
DNA barcode for identifying species in question. To use
this approach on a large scale, construction of DNA barcodes for each species will provide a key for identifying
unknown species. The eciency of such a DNA based
identication system is promising with increasing taxon
coverage of gene databases and availability of faster and
cheaper sequencing techniques. To be eective, a diagnostic
barcode must demonstrate consistent dierences among
closely related species and exhibit very limited intraspecic
variation. The objective of the present study was to develop
a key for the identication of tissue samples based on independent mitochondrial and nuclear markers.
3.1. Species identication by PCR sequencing approaches
3.1.1. Mitochondrial markers (16S rRNA and ND4 gene)
On the basis of partial 16S rDNA sequence data, all tissue samples (bualo, cow, chicken, pig, goat, frog, sh and
snake) showed a high degree of similarities to corresponding sequences in GenBank database and were perfectly
identied up to species level. Thus, the partial 16S rDNA
sequences can be used as a suitable DNA barcode for species identication and authentication of animal tissues.

669
Buffalo

89

Cow

91
100

Pig
Goat

79

Fish

83

Frog
Snake
Chicken

50

40

30

20

10

Fig. 1. Phylogenetic aliation of dierent meat specimens based on partial

sequences of 16S rDNA sequences (450 bp). The phylogenetic tree was
generated using the UPGMA method in MEGA 3.1. The bar indicates the
nucleotide dierences. The numbers at nodes indicate percent bootstrap
values considering 50% majority rule consensus with 1000 replications.

The method is versatile as it can be used as a general screen

for all vertebrate species. Secondly, the richness of the 16S
rRNA gene database also makes it an excellent molecule
of choice for identication and species authentication purposes. Phylogenetic analysis of 16S rDNA sequences by
UPGMA method indicated two major clusters A and B,
which were genetically related to each other at 50% similarity (Fig. 1). Cluster A was highly heterogeneous and was
further subdivided into three dierent subclusters. Subcluster I contained 4 phylogenetically closed specimens bualo,
cow, pig and goat that were genetically related to each other
with similarity ranging from 81% to 95%. Subcluster II contained frog and sh samples that were related to each other
at 73% similarity. Subcluster III contained only the snake
sample that was related to other samples at 4079% similarity. Cluster B contained only the chicken sample, which
showed very low degree of similarity ranging from 37% to
40% with other tissues. Unknown specimens viz. blood,
brain and viscera showed 99100% nucleotide similarity at
16S rDNA level to the leopard (Panthera pardus).
Species identication using the other mitochondrial marker viz. ND4 was much better compared to 16S rDNA.
Most of the samples showed similarity to the corresponding
sequence with much higher score values and were identied
up to species level except for the sh and snake samples.
ND4 gene sequences identied these two samples as Porichthys myriaster (toad shes) and Cylindrophis ruus (redtailed pipe snake found in Southeast Asia) respectively
because of the unavailability of ND4 gene sequences
belonging to genus Mystus cavasius and Uropletis sp.
respectively in the GenBank database (Table 2). Phylogenetic analysis of ND4 sequences based on the UPGMA
algorithm identied two major clusters A and B (Fig. 2).
Cluster A was further subdivided into two subclusters I
and II. Subcluster I included bualo, cow, pig and chicken
samples, which were genetically related at 6379% similarity. Subcluster II included only the snake sample with similarity ranging from 32% to 64% with other tissue samples.
Cluster B was further subdivided into two subclusters I and
II. Subcluster I contained only the frog sample, which
showed similarity of 3464% with the goat and sh samples,

670

Table 2
Similarities for the sequences of mt-16S rDNA, mt-ND4 and nu-actin gene
Specimen used for analysis

Gene amplied

Accession numbera

Closest match at Genbank

Accession number

Score (bit)

E-value

Bualo (Bubalus bubalis)

16S rRNA
ND4
Actin

DQ904379
DQ904397
DQ904371

Bubalus bubalis (Bovidae)

Bubalus bubalis
Bos taurus (Bovidae)

AF547270
AY488491
U02285

1045
1620
440

0.0
0.0
3e120

Cow (Bos indicus)

16S rRNA
ND4
Actin

DQ904381
DQ904390
DQ904372

Bos indicus (Bovidae)

Bos indicus breed Nellore
Bos taurus

AF492350
AY126697
BC102992

932
700
1108

0.0
0.0
0.0

100
94
98

Chicken (Gallus gallus)

16S rRNA
ND4
Actin

DQ904380
DQ904391
DQ904373

Gallus gallus breed Tibetan (Phasianinae)

Gallus gallus
Gallus gallus

DQ648776
X52392
AF012348

1110
724
458

0.0
0.0
1e125

100
97
94

Pig (Sus scrofa)

16S rRNA
ND4
Actin

DQ904382
DQ904398
DQ904374

Sus scrofa breed Large White (Suidae)

Sus scrofa breed Lanyu
Sus scrofa

AF486874
DQ518915
U16368

896
1241
446

0.0
0.0
5e122

98
96
94

Goat (Capra hircus)

16S rRNA
ND4
Actin

DQ904383
DQ904395
DQ904375

Capra hircus (Caprinae)

Capra hircus
Bos taurus

AF533441
AF533441
U02285

331
1124
416

7e88
0.0
2e113

92
100
95

Frog (Bufo melanostictus)

16S rRNA
ND4
Actin

DQ904384
DQ904392
DQ904376

Bufo melanostictus (Bufonidae)

Bufo melanostictus
Hyla japonica (Hylidae)

AY458592
AY458592
AB092520

880
2516
618

0.0
0.0
3e174

97
99
98

Fish (Mystus cavasius)

16S rRNA
ND4
Actin

DQ904389
DQ904393
DQ904378

Mystus sp. (Bagridae)

Porichthys myriaster (Batrachoididae)
Morulius calbasu (Cyprinidae)

AY458877
AP006739
AF393832

952
2664
1982

0.0
0.0
0.0

97
99
97

Snake (Uropeltis sp.)

16S rRNA
ND4
Actin

DQ904385
DQ904396
DQ904377

Uropeltis sp. (Uropeltidae)

Cylindrophis ruus (Cylindrophiidae)
Pseudonaja textilis (Elapidae)

AY701032
AB179619
AY734452

797
365
1255

0.0
2e97
0.0

96
83
99

Panthera pardus (blood specimen)

16S rRNA
ND4

DQ904386
DQ904394

Panthera pardus (Felidea)

Felis catus (Felidea)

DQ530062
U20753

733
2633

0.0
0.0

100
99

Panthera pardus (brain specimen)

16S rRNA
ND4

DQ904386
DQ904394

Panthera pardus
Felis catus

DQ530062
U20753

839
2633

0.0
0.0

100
99

Panthera pardus (viscera specimen)

16S rRNA
ND4

DQ904386
DQ904394

Panthera pardus
Felis catus

DQ530062
U20753

906
2633

0.0
0.0

99
99

Percentage similarity
99
98
95

G. Rastogi et al. / Meat Science 76 (2007) 666674

Blast results indicating the similarity of each species included in the study with closest known species in the database, with bit-scores and E-value for each mitochondrial and nuclear markers. Bracketed
denotion in front of species name indicates family name for the particular specimen tested.
a
Accession numbers generated in this study.

G. Rastogi et al. / Meat Science 76 (2007) 666674

Buffalo

100

Cow

100
87

Pig

100

Chicken
Snake
Frog
Goat

94

Fish
140

120

100

80

60

40

20

Fig. 2. Phylogenetic aliation of dierent meat specimens based on

partial sequences of NADH dehydrogenase subunit 4 gene sequences
(600 bp). The phylogenetic tree was generated using the UPGMA
method in MEGA 3.1. The bar indicates the nucleotide dierences. The
numbers at nodes indicate percent bootstrap values considering 50%
majority rule consensus with 1000 replications.

Fig. 3. Gel electrophoresis of PCR products showing amplication of a

single intron from meat specimens. M. 100 bp DNA ladder (Promega),
Lane 1. bualo, Lane 2. cow, Lane 3. goat, Lane 4. pig, Lane 5. chicken,
Lane 6. frog, Lane 7. sh, Lane 8. snake.

while sub cluster II comprised only the sh sample and was

genetically related to goat sample at 64% similarity. ND4
gene sequences retrieved from blood, brain and viscera
specimens were 100% identical and identied them closely
to cat (Felis catus) at 99% nucleotide similarity level.
3.1.2. Nuclear marker (actin gene)
The modied PCR conditions gave a single amplication product of 400 bp which was sequenced directly
(Fig. 3.), thus, eliminating the need for cloning the PCR
Buffalo

100

671

product and making the technique simple for routine analysis. Species identication using intron sequences present
between exon 6 and 7 of actin gene was not found to be satisfactory because only chicken and pig samples were identied perfectly up to species level using this nuclear marker.
The poor identication based on this gene was again attributed to non-availability of the actinintron sequences of B.
bubalis, B. indicus, C. hircus, B. melanostictus, Mystus sp.,
and Uropeltis sp. in GenBank for similarity studies. Phylogenetic analysis based on intron sequences identied two
clusters A and B at 70% similarity (Fig. 4). Cluster A
was further subdivided into three subclusters I, II, III. Subcluster I contained three samples bualo, cow and pig that
were related to each other at similarity ranging from 48
98%. Subcluster II included only the chicken sample which
showed similarity of 3256% with other samples, while sub
cluster III contained only the goat sample with 3259%
similarity with other samples. Cluster B contained three
samples frog, sh and snake that were related to each other
with similarity ranging from 83% to 89%.
3.1.2.1. Nucleotide accessions numbers. The nucleotide gene
sequences generated under this study for 16S rDNA, ND4
and actinintron genes were deposited in GenBank under
accession numbers DQ904379DQ904389, DQ904390
DQ904398 and DQ904371DQ904378 respectively.
3.1.3. Species identication by PCR ngerprinting
Both RAPD and actin ngerprinting methods allowed
dierentiation among tissue samples. RAPD ngerprinting
resulted in relatively simple DNA ngerprints from which
the species origin could be visually inferred, making the
technique especially suitable for routine analysis. The
application of actin barcoding also allowed the discrimination among dierent tissue samples. However, it gave a
higher number of bands, including a signicant number
of faint bands, which made visual interpretation a little difcult. Although the unique properties of the actin multigene family make it attractive for further authentication
studies, the low template copy number per cell is disadvantageous in terms of sensitivity, compared to more abundant
mt-DNA templates (Fairbrother et al., 1998).

Cow

100
35

Pig

3.2. RAPD ngerprinting

Chicken
Goat
Frog
100

Snake

87

Fish
30

20

10

Fig. 4. Phylogenetic aliation of dierent meat specimens based on

partial sequences of the actinintron gene sequences (350 bp). The
phylogenetic tree was generated using the UPGMA method in MEGA 3.1
software. The bar indicates the nucleotide dierences. The numbers at
nodes indicate percent bootstrap values considering 50% majority rule
consensus with 1000 replications.

The discriminating ability of RAPD-PCR assay is virtually unlimited as it is always possible to use other random
primers. In the present study, we tested ve primers
(Operon I. D. C4, D5, F4, F7 and F5) and, nally one of
them (F4) was selected for RAPD ngerprinting on the
basis of number, intensity and distribution of bands to
clearly discriminate various species. Our data clearly
showed that in each case primer F4 was able to generate
characteristic species-specic ngerprints. RAPD ngerprinting with primer F4 allowed the discrimination among
bualo, cow goat, pig and chicken tissue (Fig. 5). The analysis has been repeated twice on two dierent occasion and

672

G. Rastogi et al. / Meat Science 76 (2007) 666674

Fig. 5. RAPD analysis using the primer F4. Fingerprints were visualized
on 2% agarose gel. Lane 1. bualo, Lane 2. chicken, Lane 3. cow, Lane 4.
goat, Lane 5. pig, Lane 6. 1Kb plus DNA ladder (Invitrogen).

Table 3
Similarity matrix based on RAPD ngerprinting data showing percentage
relatedness among tissue specimens
Specimen

Cow

Chicken

Bualo

Pig

Goat

Cow
Chicken
Bualo
Pig
Goat

100
17.6
18.1
15.9
17.3

100
2.9
2.9
7.1

100
28.1
15.7

100
24.3

100

consistent results could be obtained every time (data not

shown). This was achieved by the introduction of a prolonged ramp time between annealing and extension tem-

Fig. 6. Dendrogram based on RAPD ngerprinting data displaying

results of a cluster analysis using the dice similarity coecient as a measure
of similarity among the tissue specimens analyzed.

peratures in PCR (Louie et al., 1996). Samples

corresponding to each meat tissue showed unique RAPD
patterns and showed clustering with other tissue samples
only at very low similarity ranging from 2.9% to 28.1%
(Table 3). Amplication products ranged from 300 to
2000 bp with 89 bands of varying intensity. UPGMA cluster analysis on the basis of the RAPD ngerprinting data
showed genetic relatedness among ve tissue samples under
investigation at 0.111.00 dice similarity coecient. Two
major clusters A and B were formed at 11% similarity
(SD = 0.11) but showing dierent compositions and lower
similarity level to those obtained with actin ngerprinting
(Fig. 6). Cluster A contained the chicken and cow samples,
which were related to each other with 17.6% similarity.
Another cluster B was highly heterogeneous and contained
tissue of distantly related species. Cluster B was further
subdivided into two subclusters, which were formed at
20% similarity. Subcluster I included only the goat sample
which was related to the pig and bualo samples at 24.3%
and 15.7% similarity respectively. Subcluster II included
two samples bualo and pig which were related at 28.1%
similarity. Overall, RAPD ngerprints obtained with primer F4 were clear enough to allow discrimination among
dierent tissue samples. The reproducibility and simplicity
of these proles integrated by a small but reproducible
number of bands makes the technique especially suitable
for routine analysis in food inspection programs due to
the easy interpretation of the results by visual inspection.
3.2.1. Actin ngerprinting
A major disadvantage of mt-DNA sequencing is that the
mt-genome is inherited maternally in most animals, and so
hybrids possess only the genetic signature of the maternal
parent. In cases where natural hybridization occurs mitochondrial data must be supplemented with molecular tools
based on biparentaly inherited nuclear genes. Independent
verication of such mt-DNA identications requires analysis of nuclear genetic loci, but this is technically more dicult than standard mt-DNA sequencing. Using nuclear
primers to investigate species identities in forensic studies
has the advantage that conclusions can be veried by
examining a number of independent loci. The existence of
intron size variability between exon 6 and 7 in actin gene
leads to a multiplex PCR reaction amplifying several actin
gene loci simultaneously, generating a ngerprint or
barcode characteristic of each species tested (Fig. 7).
The primers used in generating the ngerprint were
reported to produce stable patterns with no or minimum
detection of variability among individuals or breeds (Fairbrother et al., 1998; Svenson & Whiting, 2004). The discriminating ability of actin barcoding was excellent.
Amplication products ranged from 95 to 705 bp with 4
7 bands of varying intensity. UPGMA cluster analysis on
the basis of actin ngerprinting data showed genetic relatedness among 5 tissue samples under investigation at 0.24
1.00 on the dice similarity coecient scale. Two major clusters A and B were dened at 24% similarity (SD = 0.24)

G. Rastogi et al. / Meat Science 76 (2007) 666674

673

(Fig. 8). Cluster A contained pig and chicken samples,

which were related at 49% similarity and shared bands of
520 bp and 140 bp in common. Another cluster B was further subdivided into two subclusters, which were formed at
39% similarity. Subcluster I contained only the cow sample
that shared a band of 705 bp with the bualo and goat
samples and was found to have 40% and 38.4% similarity
respectively. Subcluster II included two specimens, goat
and bualo that were related at 46% similarity level. Thus,
in all cases actin barcodes were related to each other with
very weak similarity ranging from 8.2% to 49.2% (Table
4) and allowed identication of species origin of these tissues. However, more complex patterns were generated
compared to RAPD-PCR including low intensity bands,
which introduced some diculty in visual interpretation.
4. Conclusions

Fig. 7. Actin ngerprinting using the primers targeting the intron region
present between exon 6 and 7 of multiple actin genes. The ngerprints
were visualized on 2% agarose gel. Lane 1. bualo, Lane 2. chicken, Lane
3. cow, Lane 4. goat, Lane 5. pig, Lane 6. 1Kb plus DNA ladder
(Invitrogen).

In conclusion, our results indicated that mt-DNA based

markers are more suitable than nuclear markers. This superiority can be attributed to the fact that mt-DNA evolves
relatively rapidly at the sequence level because of inherent
mitochondrial ineciency to repair replication errors and
DNA damage than nuclear DNA resulting in the accumulation of dierences between closely related species (Brown,
George, & Wilson, 1979). All markers identied samples of
blood, brain and viscera belonging to family Felidae and
therefore eliminating the possibility of its human origin
as suspected by forensic authorities. Among PCR ngerprinting approaches, RAPD was found to be more accurate, producing relatively simple ngerprints from
which the species identication can be visually inferred,
making this method especially suitable in routine food
inspection programmes. The long term goal of our research
is to establish a library of reference ngerprint patterns for
the various meat species, as well as to identify the most
suitable primer sets to be used for the identication of specic meat species.
Acknowledgements

Fig. 8. Dendrogram based on actin ngerprinting data displaying results

of a cluster analysis using dice similarity coecient as a measure of
similarity among the tissue specimens analyzed.

Table 4
Similarity matrix based on actin ngerprinting data showing percentage
relatedness among tissue specimens
Specimen

Bualo

Chicken

Bualo
Chicken
Cow
Goat
Pig

100
19.9
40
46
21.8

100
13.6
39.4
49.2

Cow

Goat

We are thankful to the sta of the Regional Forensic

Laboratory, Pune, India for providing the forensic specimens. Authors are also thankful to Dr. Hemant Ghate,
Head of Department of Zoology, Modern Science College,
Pune for the phenotypic identication of specimens. The
authors wish to thank Mr. Sayed Kadri and Mr. Nanaware
for their help in sample collection. GR would like to acknowledge Council of Scientic and Industrial Research,
New Delhi, India. All authors are thankful to the anonymous reviewers for their comments and suggestions.

Pig

References
100
38.4
8.2

100
40.2

100

Aida, A. A., Che Man, Y. B., Wong, C. M. V. L., Raha, A. R., & Son, R.
(2005). Analysis of raw meats and fats of pigs using polymerase chain
reaction for Halal authentication. Meat Science, 69, 4752.

674

G. Rastogi et al. / Meat Science 76 (2007) 666674

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J.
(1990). Basic local alignment search tool. Journal of Molecular Biology,
215, 403410.
Arevalo, E., Davis, S. K., & Sites, J. W. J. (1994). Mitochondrial DNA
sequence divergence and phylogenetic relationships among 8 chromosome races of the Scleropus gramnicus complex (Phrynsomatidae) in
central Mexico. Systematic Biology, 43, 387418.
Borgo, R., Souty Grosset, C., Bouchon & Gomot, D. L. (1996). PCRRFLP analysis of mitochondrial DNA for identication of quail meat
species. Journal of Food Science, 61, 14.
Brown, J. W. (1999). The Ribonuclease P database. Nucleic Acids
Research, 27, 314.
Brown, W. M., George, M. J., & Wilson, A. C. (1979). Rapid evolution of
animal mitochondrial DNA. Proceedings of National Academy of
Sciences USA, 176, 19671971.
Calvo, J. H., Zaragoza, P., & Osta, R. (2001). Random amplied
polymorphic DNA ngerprints for identication of species in poultry
pate. Poultry Science, 80, 522524.
Cespedes, A., Garcia, T., Carrera, E., Gonzalez, I., Fernandez, A.,
Hernandez, P. E., et al. (1999). Application of polymerase chain
reaction-single strand conformational polymorphism (PCR-SSCP) to
identication of atsh species. Journal of AOAC International, 82,
903907.
Charnyi, V. I. (1967). The reaction of precipitation in agar as a method of
dierentiating the protein of phylogentically related animals. Sudan
Medicine Ekspert, 10, 415.
Fairbrother, K. S., Hopwood, A. J., Lockley, A. K., & Bardsley, R. G.
(1998). The actin multigene family and livestock speciation using the
polymerase chain reaction. Animal Biotechnology, 9, 89100.
Felsenstein, J. (1989). PHYLIP-Phylogeny Inference Package (Version
3.2). Cladistics, 5, 164166.
Franklin, R. B., Taylor, D. R., & Mills, A. L. (1999). Characterization of
microbial communities using randomly amplied polymorphic DNA
(RAPD). Journal of Microbiological Methods, 35, 225235.
Goebel, A. M., Donnelly, J. M., & Atz, M. E. (1999). PCR primers and
amplication methods for 12S ribosomal DNA, the control region,
cytochrome oxidase I, and cytochrome b in bufonids and other frogs, and
an overview of PCR primers which have amplied DNA in amphibians
successfully. Molecular Phylogenetics and Evolution, 11, 163199.
Hillis, D. M., Mable, B. K., & Moritz, C. (1996). Applications of
molecular systematics: the state of the eld and a look to the future. In
Molecular systematics (2nd ed.) (pp. 515545). Sunderland, Mass:
Sinauer Associates.
Hunt, D. J., Parkes, H. C., & Lumley, I. D. (1997). Identication of the
species of origin of raw and cooked meat products using oligonucleotide probes. Food Chemistry, 60, 437442.

Kumar, S., Tamura, K., & Nei, M. (2004). MEGA3: integrated software
for molecular evolutionary genetics analysis and sequence alignment.
Bioinformatics, 5, 150163.
Lorenzini, R. (2005). DNA forensics and the poaching of wildlife in Italy:
a case study. Forensic Science International, 153, 218221.
Louie, M., Jayaratne, P., Luchsinger, I., Devenish, J., Yao, J.,
Schlech, W., et al. (1996). Comparison of ribotyping, arbitrarily
primed PCR, and pulsed-eld gel electrophoresis for molecular
typing of Listeria monocytogenes. Journal of Clinical Microbiology,
34, 1519.
Meyer, R., Hofelein, C., Luthy, J., & Candrian, U. (1995). Polymerase
chain reaction-restriction fragment length polymorphism analysis: a
simple method for species identication in food. Journal of AOAC
International, 78, 15421551.
Palumbi, S. R., & Cipriano, F. (1998). Species identication using genetic
tools: the value of nuclear and mitochondrial gene sequences in whale
conservation. Journal of Heredity, 89, 459464.
Patterson, R. L. S. (1995). Biochemical identication of meat species. Essex,
U.K.: Elsevier Applied Science Publishers.
Rastogi, G., Bharde, A., Dharne, M., Pandav, V. S., Ghumatkar, S. V.,
Shinde, B. M., Patole, M. S., et al. (2004). Species determination and
authentication of meat samples by mitochondrial 12S rRNA sequence
analysis and conformation sensitive gel electrophoresis. Current
Science, 87, 12781281.
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning.
New York, USA: Cold Spring Harbor Laboratory Press.
Simon, C. F., Frati, A., Beckenbach, B., Crespi, H., Liu & Flook, P.
(1994). Evolution, weighting, and phylogenetic utility of mitochondrial
gene sequences and a compilation of conserved polymerase chain
reaction primers. Annals of Entomological Society of America, 87,
651701.
Svenson, G. J., & Whiting, M. F. (2004). Phylogeny of Mantodea based on
molecular data: evolution of a charismatic predator. Systematic
Entomology, 29, 359370.
Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specic gap penalties and weight
matrix choice. Nucleic Acids Research, 22, 46734680.
Welsh, J., & McClelland, M. (1990). Fingerprinting genomes using PCR
with arbitrary primers. Nucleic Acids Research, 18, 7213
7218.
Wong, K. L., Wang, J., But, P. P., & Shaw, P. C. (2004). Application of
cytochrome b DNA sequences for the authentication of endangered
snake species. Forensic Science International, 139, 4955.
Xia, X. (2000). Data analysis in molecular biology and evolution (276pp).
Boston: Kluwer Academic publishers.