PII: ELSEVIER

so309-1740(97)00104-6

Meat Science, Vol. 48, No. 314, 215-285, 1998 Q 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/98 $19.00 +O.OO

Random Amplified Polymorphic DNA (RAPD) Fingerprints for Identification of Red Meat Animal Species
M. C. Koh, C. H. Lim, S. B. Chua, S. T. Chew & S. T. W. Phang*
Veterinary Public Health Laboratory, Primary Production Singapore 619495 Department, 51 Jalan Buroh,

(Received

16 February

1997; revised version received 8 September

1997; accepted

9 September

1997)

ABSTRACT The random amplifiedpolymorphic DNA (RAPD) method was used to generatejngerprint patternsfor 10 meat species: wild boar, pig, horse, buffalo. beef, venison, dog, cat, rabbit and kangaroo. A total of 29 IO-nucleotide primers, with GC contents ranging from 5WO%. were evaluatedfor their spectjicity and eficiency. The fingerprint patterns that were generated were found in some cases to be species-spect$c, i.e. one species could be dtflerentiated from another. The advantages and disadvantages of using RAPDPCRfor the identification of red meat species are also discussed. 0 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION The problem of substitution or adulteration of costly meat with a cheaper one, whether by accident or intention, is not a new one. Determining the species which meat originated from is an integral part of food regulatory control with respect to economic fraudulence. Apart from possible economic loss, correct species identification is important to the consumer for other reasons : medical requirements of individuals who may have specific food allergies or religious dietary restrictions. Visual differentiation of similarly-pigmented meats (especially if the:. have been frozen in blocks) is almost impossible. This situation has prompted research to find methods for the detection of the origin of meat in food products. Several methods have been reported for differentiation of red meats. Among them are the Ouchterlony method based on antigen-antibody reaction (Ouchterlony, 1949; Kangethe et al., 1986), SDS-PAGE (Zerifi et al., 1992), ELISA (Martin et al., 1991b; Andrews et al., 1992) and isoelectric focusing (IEF) (King, 1984). Each of these methods have their disadvantages. For instance, the Ouchterlony method cannot distinguish between closely-related species such as wild boar and pig, cattle and buffalo, sheep and goat. The effectiveness of ELISA and SDS-PAGE is hampered by the cumbersome

*To whom correspondence

should be addressed.

Fax: 65 2650784; e-mail: Stefan_Phang@ppd. 275

gov.sg

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process of isolating species-specific proteins. IEF presupposes that the protein composition of meat is similar within species, and have differences between, for instance, muscle proteins of sheep and goat. However, even the electrophoresis patterns of serum proteins and brain proteins could be different within the same species. The present study attempts to overcome the disadvantages and ambiguities by using a rapid polymerase chain reaction (PCR)-based method known as random amplified polymorphic DNA (RAPD). This method has been shown to generate informative fingerprint patterns in a relatively short time (Williams et al., 1990; Welsh and McClelland, 1990). No prior knowledge of DNA sequences is required. Commercially-available lo-nucleotide (nt) primers are used in one single PCR program. The results shows that RAPD could be used for the discrimination of species of different red meats. Ten species of red meats were used in the study, namely: beef, pork, kangaroo, venison, wildboar, horse, buffalo, rabbit, dog and cat. We intend to establish a library of reference fingerprint patterns for the various meat species, as well as to identify the most suitable primer sets to be used for the identification of specific meat species. MATERIALS Meat samples The meat samples used in this study were collected from imported consignments by Veterinary Public Health Inspectors. Ten meat species previously characterized by IEF were selected for DNA extraction and subsequent RAPD analysis. These were beef, buffalo, kangaroo, horse, pig, wild boar, deer, cat, dog and rabbit. Samples received were visuaIly examined for general physical appearance. The samples were then rinsed, extraneous fat removed, minced and kept frozen for DNA extraction. Extreme care was taken to prevent contamination between different samples during the preparation procedures. DNA extraction Genomic DNA from the meat samples were isolated following the method of Krieg et al. (1983) with modifications. All chemicals and reagents used in this study were of molecular biology grade. Briefly, approximately 1.Og of frozen minced tissue was thawed and placed in a 15 ml sterile centrifuge tube (Costar); 3 ml of SSE buffer (0.3 M Sodium acetate, 0.5% SDS, 5 mM EDTA, pH 8.3) was added, followed by 3 ml SSE-equilibrated phenol and 3 ml chloroform/isoamyl alcohol (24: 1). The tube was then shaken vigorously for 5 min, followed by centrifugation at 3000 g for 15 min. The aqueous phase was extracted twice with chloroform/isoamyl alcohol. The aqueous phase from the final chloroform/isoamyl alcohol extraction was transferred to a new tube and the DNA was recovered by precipitation with 2 vol. of cold absolute ethanol and left to precipitate for at least 2 hr at -20C. The DNA was collected by centrifugation at 10 000 g for 15 min, washed and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8-O). The purity and concentration of the DNA were determined using GeneQuant DNA Calculator (Pharmacia, Sweden). The samples were then standardized to 500 ng ~1~ and stored in aliquots. RAPD-PCR lo-nt DNA primers used for this study were purchased from Genosys Biotechnologies (USA). The freeze-dried primers were reconstituted in TE buffer before use. The primer designations and sequences are shown in Table 1. AND METHODS

RAPD of red meat animals TABLE 1 Primers Used for RAPD-PCR of Red Meat Species Primer designation SO-01 SO-02 SO-03 so-04 50-05 SO-06 60-O 1 60-02 60-03 60-04 60-05 60-06 60-07 60-08 70-01 70-02 70-03 70-04 70-0s 80-01 80-02 80-03 80-04 80-05 80-06 80-07 80-08 80-09 80-10 % GC content 50 50 50 50 50 50 60 60 60 60 60 60 60 60 70 70 70 70 70 80 80 80 80 80 80 80 80 80 80 Sequence (S-3) GTGCAATGAG CAATGCGTCT AGGATACGTG TCCCMTAGC CGGATAACTG AGGTTCTAGC CGCAGTACTC GTCCTACTCG CTACACAGGC GTCC?TAGCG GTCCTCAACG CTACTACCGC GAGTCACTCG GTCCTCAGTG CATCCCGAAC CAGGGTCGAC ACGGTGCCTG CGCATTCCGC GAGATCCGCG GCACCCGACG CGCCCAAGCC CCATGGCGCC CGCCCGATCC ACCCCAGCCG GCACGGAGGG GCACGCCGGA CGCCAGCAGC GCACGGTGGG CGCCCTGGTC

211

The PCR reaction was carried out in a total volume of 25 ~1 using 200 ng genomic DNA as template. The reaction mixture contained 2.5 ~1 of 10X PCR buffer (Pharmacia, supplied together with Taq polymerase), 2.0 mM MgC12, 20 pmol lo-nt primers, 0.2 mM dNTP (Pharmacia) and 1U Taq Polymerase (Pharmacia). The amplification was carried out in a thermal cycler (Perkin Elmer Cetus). The samples were denatured at 96C for 5 min, followed by 35 cycles of 94C (1 min), 32C (1 min) and 72C (2 min). The RAPDPCR products were electrophoresed in 1% agarose gels containing 1-O,ug ml- ethidium bromide and 1X TBE (containing O-9M Tris-OH, 0.5 M boric acid and 20 mM EDTA) as running buffer. The gels were visualized and photographed under UV using a Polaroid 322 camera and T667 film.
RAPD analysis

In this study, RAPD profiles were analysed de visu and a simple scoring system of + (denoting fingerprint patterns of at least two distinct DNA bands generated) and - (no fingerprint patterns of at least two distinct DNA bands) was used, provided that these bands are reproducible within the particular species under the same controlled conditions.

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Reproducibility was checked by subjecting 10 different animals from each species to the same PCR and electrophoresis conditions. RESULTS AND DISCUSSION

High molecular weight DNA was successfully extracted from all samples. The ratio of Aza: Azs,, ranged from 1.6 to 2.0, which was sufficiently pure for PCR reactions (Sambrook et al., 1989). A total of 29 primers with varying GC contents were evaluated. These include 50% GC (6 different sequences), 60% (8 sequences), 70% (5 sequences) and 80% (10 sequences). Only one PCR program was used. This was to standardize the evaluation of the primers. The results with respect to primer efficiency and species-specificity are summarized in Tables 2-5. These results were judged as provisonal observations only, as the PCR was carried out under one set of non-optimised cycling conditions. Generally, experiments
TABLE 2

Efficiency of 80% GC Primer Series

Primer designation Meat species 80-01 80-02 80-03 80-04 80-05 80-06 80-07 80-08 80-09 80-10

Wild boar Pig Horse Kangaroo Buffalo Beef Venison Dog Cat Rabbit

+ + + + + + + + + -

+ + + + + + + + + -

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + _ + + + + -

+ + + + + -

+ + + + + + + + + +

+ + + + + i+ + + +

+ = Fingerprint patterns of at least two distinct generated. - = No fingerprint patterns of at least two bands generated.
TABLE 3

Efficiency of 70% GC Primer Series

Primer designation Meat species 70-01 + + + k _ _ 70-02 _ _ _ _ 70-03 i-I+ + + + + + + + 70-04 i+ + + + + + + + + 70-05 + -

Wild boar Pig Horse Kangaroo Buffalo Beef Venison Dog Cat Rabbit

+ = Fingerprint patterns of at least two distinct generated. - = No fingerprint patterns of at least two bands generated.

RAPD of red meat animals TABLE 4 Efficiency of 60% GC Primer Series Primer designation Meat species Wild boar Pig Horse Kangaroo Buffalo Beef Venison Dog Cat Rabbit 60-01 60-02 60-03 60-04 60-05 60-06 60-07

219

60-08

+ + + + + + + -

+ + -

+ + + + + + + + +

_ + _ -

+ + + + -

+ + + + -

+ = Fingerprint patterns of at least two distinct generated. - = No fingerprint patterns of at least two bands generated. TABLE 5 of 50% GC Primer Series Primer designation Meat species Wild boar Pig Horse Kangaroo Buffalo Beef Venison Dog Cat Rabbit 50-01 + + + + + + + + + + 50-02 + _ SO-03 + + + 50-04 + + 50-05 _ _ _ _ + + SO-06 + + + + + + + + + +

Efficiency

+ = Fingerprint patterns of at least two distinct generated. - = No fingerprint patterns of at least two bands generated.

with the 80% GC primers showed clear fingerprint patterns, compared to the other primers. Eight out of the ten 80% GC series showed fingerprint patterns that could be used to discriminate among several species, although not all of the species (Table 2). We are especially concerned that the primers used should be able to discriminate unequivocally between beef vs kangaroo vs buffalo vs horse, as the latter three species are used frequently to substitute for beef in meat products (Keenan and Shaklee, 1985). Along the same line of thought, we also needed to discriminate between wild boar and pig. Figure l(a) and (b) show the typical fingerprint patterns for all the 10 meat species studied, using two of the 80% GC primer series, 80-04 and 80-10. Primer 80-04 showed unique fingerprint patterns for at least 9 of the 10 species investigated, but the discriminatory potential of primer 80-10 is suspect, although this primer was able to generate fingerprint patterns for all the 10 species tested. Table 3 summarizes the results observed for the 70% GC primer series. Although primers 70-03 and 70-04 [Fig. 2(a) and (b)] showed fingerprints for all the species, the patterns for the discrimination of beef vs buffalo (primer 70-03) and between wild boar and pig (primer 70-04) were not convincing enough.

280 M 1 2 3

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10

(4
M 1 2 3 4 5 6 7 8 9 10

Fig. 1. (a) Results of RAPD-PCR fingerprinting by primer 80-04, and (b) by primer 80-10. Lanes : M, lOO-bp DNA markers (Pharmacia), 1, wild boar; 2, pig; 3, horse; 4, kangaroo; 5, buffalo; 6, beef; 7, venison; 8, dog; 9, cat; 10, rabbit. Results from the 60% GC series are summarized in Table 4. Primers 60-01 and 60-03 showed good fingerprint patterns [Fig. 3(a) and (b)], but repeatedly did not show any amplification with dog DNA. In addition, primer 60-01 did not amplify buffalo DNA. Three out of six 50% primers tested showed relatively good amplification patterns for most of the species. Two of the pattern sets generated by primers 50-01 and 50-06 are shown in Fig. 4(a) and (b). However, although these primers could generate patterns for various species tested, these could not discriminate very well between the 10 species. For example, primer 50-01 could not discriminate between beef vs buffalo vs kangaroo; primer 50-06 could not discriminate between wild boar and pig. Figure 5(a) and (b) shows the fingerprint patterns generated when samples of DNA from 10 different animals from within each species of beef and pork were tested. Similar

RAPD

of red meat animals

281 8 910

12

(a)
Ml 2 3 4 5 6 7 8 910

(b)
Fig. 2. (a) Results of RAP&PCR fingerprinting by primer 70-03, and (b) by primer 70-04. Lanes : M, lOO-bp DNA markers (Pharmacia), 1, wild boar; 2, pig; 3, horse; 4, kangaroo; 5, buffalo; 6, beef; 7, venison; 8, dog; 9, cat; 10, rabbit.

results were also observed for other species (data not shown). The bovine DNA patterns in Lanes 2 and 7 of Fig. 5(a) seemed different from the other samples, and in Fig. 5(b), porcine DNA in Lane 1 also showed certain differences. This phenomenon could possibly be due to reasons genetic (intra-species polymorphism) or physical (degraded DNA). In some cases where no reaction was seen with a particular primer, repeats were carried out. This was to rule out the possibility that the PCR reagents (e.g. Taq polymerase, primers) on that particular day did not work, although this was remote as all the other

282 M 1 2 3

M. C. Koh et al. 4 5 6 7 8 9 10

(4
M 1 2 3 4 5 6 7 8 9 10

(W
Fig. 3. (a) Results of RAPD-PCR
M, lOO-bp DNA markers fingerprinting by primer 60-01, and (b) by primer 60-03. Lanes : (Pharmacia), 1, wild boar; 2, pig; 3, horse; 4, kangaroo; 5, buffalo; 6, beef; 7, venison; 8, dog; 9, cat; 10, rabbit.

on the same gel used the same PCR cocktail and underwent the same PCR procedure. The discriminating ability of the RAPD method is virtually unlimited as it is always possible to use other random primers. The procedure was also simpler and more rapid than other molecular methods such as restriction enzyme analysis or pulsed field gel electrophoresis. In this study, a total of 29 primers of varying GC contents ranging from 50 to 80% were tested. This is because some workers have found that a change of one base pair in the samples

RAPD Ml2345678910

of red meat animals

283

(a) M
I
2345678 9 10

03
Fig. 4. (a) Results of RAPISPCR fingerprinting by primer 50-01, and (b) by primer SO-06. Lanes : M, lOO-bp DNA markers (Pharmacia), 1, wild boar; 2, pig; 3, horse; 4, kangaroo; 5, buffalo; 6, beef; 7, venison; 8, dog; 9, cat; 10, rabbit. target sequence of a genome may result in a completely different RAPD profile (Williams et al., 1990).Since each lo-bp oligonucleotide primer only covers a very limited part of the genome, important differences located on non-amplified regions could be missed. In the event of similar profiles obtained from two different species (especially closely-related species) using a particular primer, this would lead to a false conclusion that the two species are the same. Thus, it is important that a series of primers be used for any sample to be tested. RAPD-PCR fingerprints have been successfully used in species identification of plants, bacteria and animals (Welsh and McClelland, 1990; Williams et al., 1990; Martin et al., 1991~; Akopyanz et al., 1992; Riedy et al., 1992). This study used only one cycling

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Ml

910

(4
M
1

10

(b)
Fig. 5. Results of RAPD-PCR fingerprinting by primer 80-10, showing the reproducibility of the procedure, of (a) beef DNA and (b) pork DNA. Lanes: M, lOO-bp DNA markers (Pharmacia), l-10,

beef (a) and l-10, pork (b). program, using IO-nt primers with GC contents ranging from 50-80%. Although many of the primers tested did not show products, nevertheless it was felt that these cases were such because the RAPD-PCR conditions were not optimized. Work is on-going in the authors laboratory to optimize the RAPD conditions. The main advantage of RAPD is that the technique usually generates some products which can be seen as DNA fingerprints on gel electrophoresis. However, as this study has shown, the main disadvantage is that the reproducibility of the patterns are difficult to

RAPD

of red meat animals

28.5

achieve due to various factors such as cycling conditions or in&a-species polymorphisms. Thus, RAPD for species identification is likely to be used as a rapid, qualitative way for meat speciation, and known standards must be run together each time a sample is tested.

ACKNOWLEDGEMENTS The authors would like to thank the Director of Primary Production Department for his permission for this research paper to be published. We would also like to thank the Inspectors from the Food Inspection Service for their assistance in sample collection.

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37, 157-162.

Keenan, C. P. and Shaklee, J. B. (1985) Electrophoretic identification of raw and cooked fish fillets and other marine products. Food Technology in Australia 37, 117-128. King, N. L. (1984) Species identification of cooked meats by enzyme staining of isoelectricfocussing gels. Meat Science 11, 59-64. Krieg, P., Amtmann, E. and Sauer, G. (1983) The simultaneous extraction of high molecular weight DNA and RNA from solid tumours. Analytical Biochemistry 134, 288-294. Martin, R., Wardale, R. J., Jones, S. J., Hernandez, P. E. and Patterson, R. L. S. (1991a) Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork. Meat Science 30, 23-3 1. Martin, G. B., Williams, J. G. K. and Tanksley, S. D. (1991b) Rapid identification of markers linked to a pseudomonas resistance gene in tomato using random primers and near-isogenic lines.
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Riedy, M. F., Hamilton III, J. and Aquadro, C. F. (1992) Excess of non-parental bands in offspring from known primate pedigrees assayed using RAPD PCR. Nucleic Acids Research 20, 918. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbour Laboratory Press, New York. Welsh, J. and McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers.
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Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V. (1990) DNA polymorphisms amplified by arbitrary primers useful as genetic markers. Nucleic Acids
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Zerifi, A., Labie, C. and Bernard, G. (1992) SDS-PAGE technique for the species identification cooked meat. Fleischwirtsch International 1. 54-56.

of