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Meat Science 51 (1999) 143148

A quick and simple method for the identication of meat species


and meat products by PCR assay
T. Matsunaga a, K. Chikuni b*, R. Tanabe b, S. Muroya b, K. Shibata a, J. Yamada a,
Y. Shinmura a
a
Japan Meat Processors Association, Ebisu 1-5-6 Shibuya-ku, Tokyo 150, Japan
National Institute of Animal Industry, Tsukuba Norindanchi, PO Box 5, Ibaraki 305, Japan

Received 28 August 1997; received in revised form 30 June 1998; accepted 2 July 1998

Abstract
The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw
materials for products. By mixing seven primers in appropriate ratios, species-specic DNA fragments could be identied by only
one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and
reverse primers on species-specic DNA sequences for each species. PCR primers were designed to give dierent length fragments
from the six meats. The products showed species-specic DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken,
cattle, sheep, pig and horse meats, respectively. Identication is possible by electrophoresis of PCR products. Cattle, pig, chicken,
sheep and goat fragments were amplied from cooked meat heated at 100 or 120 C for 30 min, but horse DNA fragments could not
be detected from the 120 C sample. Detection limits of the DNA samples were 0.25 ng for all meats. # 1998 Elsevier Science Ltd.
All rights reserved.
Keywords: PCR; Meat species identication

1. Introduction
Accurate analytical methods are indispensable for the
labeling of meat products, and that requires simple and
fast procedures. DNA hybridization (Baur et al., 1987;
Chikuni et al., 1990; Winter et al., 1990; Ebbehj and
Thomsen, 1991a, b) and PCR methods (Chikuni et al.,
1994a, b; Fei et al., 1996) have been used for identication of meats and meat products. DNA hybridization
methods are complicated and generally inadequate, but
PCR easily amplies target regions of template DNA in
a much shorter time (Saiki et al., 1985), and thus is suitable for meat identication. Chikuni et al. (1994a, b)
distinguished sheep, goat and cattle meats using a satellite DNA sequence and eight mammals and ve birds
using the cytochrome b sequence. That method consisted of PCR amplication followed by restriction
digestions, thus the procedures for mixed meats or meat
products were complicated. Fei et al. (1996) designed
multiplex PCR primers based on mitochondrial D-loop
* Corresponding author. E-mail: chikunc@niai.arc.go.jp

DNA sequences and identied cattle, pig and chicken


meats. In the present study, the authors developed a
simple method using multiplex PCR for simultaneous
identication of six meats.
2. Methods
2.1. DNA extraction
All meat samples were obtained from commercial
sources. DNA was prepared from cattle, pig, chicken,
sheep, goat and horse meats as described by Sambrook
et al. (1989). DNA/RNA mixtures were extracted in 20
vol of 100 mM TrisHCl, (pH 9.0) containing 100 mM
NaCl, 5 mM EDTA and 1% SDS, for 30 min at the
room temperature. The DNA/RNA solutions were
extracted with an equal volume of phenol/chloroform/
isoamyl-alcohol (25:24:1), and then with an equal
volume of chloroform. RNA in the DNA/RNA solution was degraded by 100 mg ml1 Ribonuclease A
(Sigma) for 1 h at 37 C. DNA was extracted twice with
an equal volume of phenol/chloroform/isoamyl-alcohol

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T. Matsunaga et al./Meat Science 51 (1999) 143148
Fig. 1. Nucleotide sequences of the primers and target region on cytochrome b gene. Open boxes indicate the common forward primer SIM and complementary sequences of species-specic reverse
primer. Dots and closed boxes indicate identical and dierent nucleotides to the primer sequences, respectively.

T. Matsunaga et al./Meat Science 51 (1999) 143148

and once with an equal volume of chloroform. DNA


concentrated by ethanol precipitation was dissolved in
10 mM TrisHCl, (pH 7.4) 0.1 mM EDTA for use as
the PCR template. DNA concentration was determined
based on absorbance at 260 nm. Each piece of 25 g from
cattle, pig, chicken, sheep, goat and horse meats was
heated for 30 min at 100 or 120 C. The DNA/RNA
mixture was extracted from 500 mg of heated meat with
10ml of 100 mM TrisHCl, (pH 9.0) containing 1%
SDS and 5 mM DTT for 5 min at 95 C. The DNA
solution was puried by phenol/chloroform/isoamylalcohol extraction.
2.2. Polymerase chain reaction (PCR)
PCR amplication was conducted in 50 ml of 10 mM
TrisHCl, (pH 8.3) containing 50 mM KCl, 1.5 mM
MgCl2, 200 mM dNTP mix, primer mix (460 pmol
each), 250 ng template DNA and 1.25 unit Taq DNA
polymerase (PerkinElmer). Oligonucleotide primers
were the common forward primer SIM (50 -GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA30 ) and reverse primers [goat primer G (50 -CTCGACAAATGTGAGTTACAGAGGGA-30 ), chicken primer
C (50 -AAGATACAGATGAAGAAGAATGAGGCG30 ), cattle primer B (50 -CTAGAAAAGTGTAAGACCCGTAATATAAG-30 ), sheep primer S (50 -CTATGAATGCTGTGGCTATTGTCGCA-30 ), pig primer P
(50 -GCTGATAGTAGATTTGTGATGACCGTA-30 ),
and horse primer H (50 -CTCAGATTCACTCGACGAGGGTAGTA-30 )] (Fig. 1) that were designed from
published sequences of cattle, pig, chicken, sheep, goat
and horse mitochondial cytochrome b genes (Anderson
et al., 1982; Irwin et al., 1991; Desjardins and Morais,
1990). These primers were mixed in the ratio of
1:0.2:3:0.6:3:0.6:2 for SIM:G:C:B:S:P:H, and used together for the multiplex PCRs of this study (the ratio 1
means 20 pmol primer/50 ml PCR solution). Thirty-ve
cycles of amplication were run using a GeneAmp PCR
System 2400 (PerkinElmer) as follows: denaturation at
94 C for 0.5 min, annealing at 60 C for 0.5 min, and
extension at 72 C for 0.5 min. Following amplication,
8 ml PCR solution was electrophoresed on 4% NuSieve
GTG agarose gel (FMC) for 30 min at 100 V in TAE
buer (40 mM Trisacetate, 1 mM EDTA, pH 8.0) and
then stained with ethidium bromide (0.5 mg ml1) for
1 h.

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on 38 bp sequence. Because each 1% mismatching of


bases in a double-stranded (ds) DNA reduces melting
temperature (Tm) by 11.5 C (Sambrook et al., 1989),
the mismatches of SIM would decrease the Tm of primer-template dsDNA about 1015 C. Thus, SIM was
designed as longer than species-specic primers that
were 2629 nucleotides long. The reverse primers were
designed on species-specic regions. The primers were
expected to amplify target sequences at the same eciency in multiplex PCR. Fig. 2 shows 4% agarose gel
electrophoresis of PCR products amplied from the six
species. PCR products from goat, chicken, cattle, sheep,
pig and horse DNAs were single DNA fragments of
157, 227, 274, 331, 398 and 439 bp, respectively. The six
meats could thus be identied based on the length of
PCR products with no cross reaction.
3.2. Identication of cooked meat
Cooked samples were obtained from the six meats
heated for 30 min at 100 or 120 C, and DNA extracted
from 500 mg of the meats was used as a template for
PCR. The PCR products were analyzed by 4% agarose
gel electophoresis. Fig. 3 shows the species-specic
DNA fragments to have been amplied from the
cooked meat heated at 100 and 120 C except for horse
meat cooked at 120 C. The cattle fragment was amplied from 120 C sample, but the signal was weaker than
the other species.

3. Results
3.1. Meat identication by species-specic primers
Fig. 1 shows primer regions on cytochrome b
sequences of the six species. The common forward primer SIM mismatches with the six species at 35 bases

Fig. 2. Agarose gel electrophoresis of PCR product amplied from the


six meats. G, goat; C, chicken; B, cattle; S, sheep; P, pig; H, horse. M
is a molecular marker, f174/Hincdigest.

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T. Matsunaga et al./Meat Science 51 (1999) 143148

Fig. 3. Agarose gel electrophoresis of PCR products amplied from cooked meat. G, goat; C, chicken; B, cattle; S, sheep; P, pig; H, horse. Lane ()
is a PCR product amplied from a reaction solution without a template DNA. M is a molecular marker, f174/Hincdigest.

3.3. Semi-quantication of mixed DNA


DNAs extracted from cattle and pig meats were
mixed for use as templates in the ratios of 88:12, 75:25,
50:50, 25:75 and 12:88. Fig. 4 shows the bands of cattle
and pig-specic fragments of 274 and 398 bp, along with
the relationships between template DNA amounts and
band intensity. In lane 4 (50:50), two bands from cattle
and pig DNAs indicated similar amounts of PCR pro-

ducts. When pig DNA increased, from lanes 26, the


band of 398 bp fragment became intense and that of 274
bp fragment faint.
3.4. Detection limits of DNA samples
Fig. 5 shows the results of PCR amplication from
mixed DNA templates of 25, 2.5, 0.25, 0.025 and 0.0025
ng each. Low molecular bands in all lanes in many cases
were probably primerdimers produced from seven primers during PCR. Lanes 13 show six bands corresponding to the six species, thus the detection limits was
0.25 ng for all meat species.
4. Discussion

Fig. 4. Agarose gel electrophoresis of PCR products amplied from


cattle and pig DNA mixture. Lanes 1,(100:0); 2,(88:12); 3,(75:25);
4,(50:50); 5,(25:75); 6,(12:88); 7,(0:100). M is molecular marker,
f174/Hincdigest.

The aim of this study was to develop a simple method


for simultaneous identication of multiple meat species.
Multiplex PCR, in which many primers are used together for amplication of multiple target regions, is a
hopeful technique for this purpose. The design of primers is very important on multiplex PCR techniques,
because primer specicity and Tm are more critical than
conventional PCRs. Ratios of mismatching in this study
were more than 15% between a species-specic primer
and the other species sequences except a pair of sheep
primer and goat template. The mismatches more than
15% decrease Tm more than 15 C, and that make
reverse primers anneal only to the species-specic
sequence in multiplex PCR. The sheep primer S mismatched with goat DNA at only two nucleotides, however, 30 end mismatching was fatal for PCR
amplication and resulted in no sheep band from a goat

T. Matsunaga et al./Meat Science 51 (1999) 143148

Fig. 5. Agarose gel electrophoresis of PCR products amplied from


0.0025, 0.025, 0.25, 2.5 and 25 ng from DNA of the six meats. Lanes 1,
2, 3, 4 and 5 are 25, 2.5, 0.25, 0.025 and 0.0025 ng of six meats DNA,
respectively. Lane 6 is a PCR product amplied from the reaction
solution without template DNA. M is molecular marker, f174/
Hincdigest.

template (Fig. 2). The other primers were also designed


to mismatch with dierent species at 30 end or next
nucleotides (Fig. 1).
Primer specicity in the entire DNA of a target species was examined by conventional PCR using a pair of
SIM and an each species-specic primer. The size of
PCR products was as expected with no additional fragment from a target species (data not shown). This result
showed that the species-specic primers amplied only
one size fragment from a target species. Primer specicity to the other species was examined by multiplex
PCR using the same primer mixture in the method. Fig.
2 showed that multiplex PCR resulted in a single band
of target size from one meat species and no fragment
produced by non-specic amplication.
The primers were designed to amplify target sequences of the six species at similar eciency. Fig. 6 shows,
however, multiplex PCRs using equal amount mixture
of the primers did not result in equal signals from the six
species (lanes 1,2). In general, quantitative PCR is dicult because of unequal eciency of amplication.
Amplication eciency is aected by the dierence of
primer sequences. The common primer SIM was
designed to be shared by the six species, therefore,
amplication eciency of PCR was aected by only

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Fig. 6. Agarose gel electrophoresis of PCR products amplied from


DNA of the six meats. Lanes 1, 12.5 ng; 2, 25 ng; 3, 12.5 ng; 4, 25 ng.
Lanes 1 and 2 are the products by mixed primers of the equal concentration. Lanes 3 and 4 are of the changed concentration. M is
molecular marker, f174/Hincdigest.

reverse primers. A little dierence of Tm among the


reverse primers would aect the eciency. In order to
control the eciency, the ratio of the primers was
changed according to the results of preliminary experiments. Lanes 3 and 4 in Fig. 6 showed that all bands
from the six species were detectable by using an appropriate ratio of primer mixture and Fig. 4 showed a possibility of semi-quantitative analysis for beef and pork
mixture. However, quantitative amplication was found
to be unsuccessful as for the other species. Fig. 5
showed dierent signal intensity from the same concentration of DNA of the six species.
Multiplex PCRs detected meat species prepared at
high temperature (Fig. 3). Cooked meat DNAs (except
horse) were amplied from all samples heated at all
temperatures, but no horse DNA fragment could be
detected at 120 C. Because 439 bp horse DNA was the
longest fragment, targeted horse DNA fragment would
be aected more by heat than other DNA fragments at
high temperature. The primer for horse should thus be
designed to amplify a shorter fragment.
PCR is quite useful for routine analysis of meat species identication, being quick and sensitive. By the
present method, the six meats could all be identied at
the same time more easily and sensitive than usual
methods. The analytical conditions must be improved
for quantitative dierentiation.

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T. Matsunaga et al./Meat Science 51 (1999) 143148

References
Anderson, S., de Bruijin, M.H.L., Coulson, A.R., Eperon, I.C., Sanger, F., Young, I.G., 1982. Complete sequence of cattle mitochondrial DNA. Conserved features of the mammalian mitochondrial
genome. Journal of Molecular Biology 156, 683717.
Baur, C., Teifel-Greiding, J., Leibhardt, E., 1987. Spezizierung hitzedenaturierter Fleischproben durch DNA-Analyse. Archiv fur
Lebensmittelhygiene 38, 172174.
Chikuni, K., Ozutsumi, K., Koishikawa, T., Kato, S., 1990. Species
identication of cooked meats by DNA hybridization assay. Meat
Science 27, 119128.
Chikuni, K., Tabata, T., Kosugiyama, M., Monma, M., Saito, M.,
1994a. Polymerase chain reaction assay for detection of sheep and
goat meats. Meat Science 37, 337345.
Chikuni, K., Tabata, T., Saito, M., Monma, M., 1994b. Sequencing of
mitochondrial cytochrome b genes for the identication of meat
species. Animal Science and Technology (Jpn) 65, 571579.
Desjardins, P., Morais, R., 1990. Sequence and gene organization of
the chicken mitochondrial genome: A novel gene order in higher
vertebrates. Journal of Molecular Biology 212, 599634.

Ebbehj, K.F., Thomsen, P.D., 1991a. Species dierentiation of heated


meat products by DNA hybridization. Meat Science 30, 221234.
Ebbehj, K.F., Thomsen, P.D., 1991b. Dierentiation of closely related species by DNA hybridization. Meat Science 30, 359366.
Fei, S., Okayama, T., Yamanoue, M., Nishikawa, I., Mannen, H.,
Tsuji, S., 1996. Species identication of meats and meat products by
PCR. Animal Science and Technology, (Jpn) 67, 900905.
Irwin, D.M., Kocher, T.D., Wilson, A.C., 1991. Evolution of the
cytochrome b gene of mammals. Journal of Molecular Evolution 32,
128144.
Saiki, R.K., Schart, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich,
H.A., Arnheim, N., 1985. Enzymatic amplication of b-globin
genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia. Science 230, 13501354.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A
Laboratory Manual, 2nd Edition. Cold Spring Harbour Laboratory
Press, New York.
Winter, A.K., Thomsen, P.D., Davies, W., 1990. A comparison of
DNA-hybridization, immunodiusion, countercurrent immunoelectrophoresis and isoelectric focusing for detecting the admixture of
pork to beef. Meat Science 27, 7585.