Beruflich Dokumente
Kultur Dokumente
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
0309-1740/98/$see front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0309 -1 740(98)00112 -0
144
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.
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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
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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.
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