Food Control 20 (2009) 366370

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species
Weibin Bai a,1, Wentao Xu a,b,1, Kunlun Huang a,b,1, Yanfang Yuan a, Sishuo Cao a, Yunbo Luo a,*
a b

Laboratory of food safety and molecular biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Supervision and Testing Center of Agricultural Products Quality, Ministry of Agriculture, Beijing 100083, China

a r t i c l e

i n f o

a b s t r a c t
A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 50 -end of species-specic reverse primers which matched with the species-specic DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specic DNA fragments amplied by CP-M-PCR method still appeared until the concentration of species-specic primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplied the PCR reaction system and conquered the disparate amplied efciency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identication of other meat species and their products. 2008 Published by Elsevier Ltd.

Article history: Received 22 September 2007 Received in revised form 12 May 2008 Accepted 20 May 2008

Keywords: Multiplex PCR Meat species Detection Common primer

1. Introduction Chicken, beef, pork and horse meat are nutrient abundant foods with the high protein and many essential amino acids, which can enhance the ability of human immunity. However, consumers are afraid for the meat food because of the spread of avian inuenza virus (Mo, Brugh, Fletcher, Rowland, & Swayne, 1997; Perkins & Swayne, 2001; Swayne & Beck, 2005), bovine spongiform encephalopathy (BSE) (Wells et al., 1987) and foot and mouth disease (FMD) (Dunn & Donaldson, 1997; Sellers & Gloster, 2007; Swayne & Beck, 2005) in chicken, cattle, pig, respectively. Species adulteration (Qinchun & Peggy, 2007) is also a severe question, which not only relates to imported products, but also has an impact on restaurants and at the retail level where the substitution is easy to conceal. For example, horse meat was adulterated in beef without corresponding label. Meat species adulteration not only constitutes economic fraud, thereby beating consumers trust in the meat food, but also relates to those with religious taboos, moral aversions, or allergies to particular meat species (Kesmen, Sahin, & Yetim, 2007). As a result, it is indispensable that meat products sold for consumers must be accurately labeled as to the species they contain. The identity of meat species in meat products to be accurately labeled is the requirement of food labeling regulations. However, accurate identication of the origin of meat species has a consider* Corresponding author. Tel./fax: +86 10 6273 6479. E-mail address: lyb@cau.edu.cn (Y. Luo). 1 These three authors contribute equally. 0956-7135/$ - see front matter 2008 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2008.05.021

able challenge for food inspectors, animal feed analysts, game enforcement authorities and individuals seeking to comply with certain religious regulations. Consumers need high quality products that are labeled honestly in order to assure meat safety. Therefore, there has been a demand for a fast and accurately applicable meat species identication system. Nowadays, numerous analytical methods have been developed based on protein and DNA analysis. Methods based on the protein fractions use techniques such as electrophoretic (Yman & Sandberg, 1987), chromatographic (Amstrong & Leach, 1992) and immunological techniques (Chi-Chung et al., 2006; Hsieh, Sheu, & Bridgman, 1998; Kangethe, Gathuma, and Landqvist, 1986). Unfortunately these methods are not sensitive in processed materials to differentiate closely related species, time-consuming, inadequate and/or expensive. Advances in DNA technology have led to the rapid development of alternative approaches for species identication. Recently, applications of polymerase chain reaction (PCR) in food analysis have been increased because of their simplicity, speediness and specicity (Chikuni, Tabata, Kosugiyama, Monma, & Saito, 1994; Kesmen, 2005; Lahiff et al., 2001; Matsunaga et al., 1999; Saiki et al., 1985). The methods currently used in meat species identication include general PCR amplication, followed by complementary techniques such as RAPD-PCR (Martinez & Yman, 1998), PCR-RFLP (Bartlett & Davidson, 1992; Brodmann & Moor, 2003; Partis et al., 2000; Wolf, Rentsch, & Hubner, 1999) and real time uorescence PCR (Brodmann & Moor, 2003). However, these methods have such an inferior reproducibility or low efciency of simultaneous detection for all kinds of meat species.

W. Bai et al. / Food Control 20 (2009) 366370 Table 1 The sequence of oligonucleotide primers Primer name CF Chicken Cattle Pig Horse Chicken-R Cattle-R Pig-R Horse-R CP Sequence 5 -gacctcccagctccatcaaacatctcatcttgatgaaa-3 50 -cagatgaagaagaatgaggcg-30 50 -ctagaaaagtgtaagacccgtaatataag-30 50 -tgatagtagatttgtgatgaccg-30 50 -cagattcactcgacgagggt-30 50 -ccttccttccttcccccccagatgaagaagaatgaggcg-30 50 -ccttccttccttccccccctagaaaagtgtaagacccgtaatataag-30 50 -ccttccttccttcccccctgatagtagatttgtgatgaccg -30 50 -ccttccttccttcccccccagattcactcgacgagggt -30 50 -ccttccttccttcccccc-30
0 0

367

Product size (bp) 221 274 394 433 239 292 412 451

References Matsunaga et al. (1999) This study This study This study This study This study This study This study This study This study

Multiplex PCR (Dalmasso et al., 2003; Fei et al., 1996; Matsunaga et al., 1999) was a new method that could simultaneously amplify template mixture and decrease the detection cost, conquering the weakness of single PCR detecting only one template once. However, multiplex PCR method had several disadvantages such as complex system with many primers, low amplied efciency and no identical efciency on different templates, which restricted the commercial application in detecting meat species. In the present study, a novel common primer multiplex PCR method named as CP-M-PCR has been developed and applied for simultaneous identication of four kinds of meats (chicken, cattle, pig and horse). 2. Methods 2.1. DNA extraction All meat samples were obtained from commercial sources. DNA was prepared from chicken, beef, pork and horse meats as described by Sambrook, Fritsch, and Maniatis (1989). DNA/RNA mixtures were extracted in 10 mL of TESR (pH 8.0), containing 50 mM TrisHCl, 50 mM NaCl, 5 mM EDTA, 1% SDS and RNA enzyme, for 3 h at 37 C. The DNA/RNA solutions were extracted twice with an equal volume of phenol/chloroform/isoamyl-alcohol (25:24:1), and then once with an equal volume of chloroform/isoamyl-alcohol (24:1). DNA concentrated by ethanol precipitation was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA) for use as the PCR template. DNA templates were quantied with the pico-green dsDNA quantication kit (Molecular Probes, Leiden, Netherlands). Fluorescence was detected using the FL800 microplate uorescence reader (Bio-Tek Instruments Inc., Winooski, Vermont, USA) and analyzed by KC4 software (2000). The number of molecules was measured according to the DNA quantity and DNA average size. 2.2. Novel common primer multiplex PCR (CP-M-PCR) In this approach, Common-F primer was designed on a conserved DNA sequence of chicken, cattle, pig and horse mitochondrial cytochrome b gene (Matsunaga et al., 1999). Both Speciesspecial reverse primers (Chicken, Cattle, Pig and Horse) and reverse primers (Chicken-R, Cattle-R, Pig-R, Horse-R) were designed on species-specic DNA sequences for each species. Reverse primers contained the species-special reverse primer at the 30 -end (in blue2) and the common adapter at the 50 -end (in red), respectively. The sequence of common primer (CP) (in red) designed was the same as the common adapter to be tagged at 50 -end respectively the complementary sequence of species-special reverse primers in CP-M-PCR. Table 1 showed the sequence of oligonucleotide

primers. All primer sets were investigated of their suitable annealing temperature by using the Biometra PCR System (TGRADIEDT). Fig. 1 showed the amplied theory of CP-M-PCR method. CP-MPCR amplication was conducted in 30 lL of 10 mM Tris-HCl (pH 8.0), containing 50 mM KCl, 1.5 mM MgCl2, dNTP mix (50 lM each), primer mix (15 pmol each), 2 unit Taq DNA polymerase (Takara) and appropriate template DNA. Multiplex PCR amplication was performed by adding mixture of new primers designed in the different ratios, and performed an optimized annealing temperature and multiplex PCR step. Following amplication, 12 ll PCR solution was electrophoresed on 2.5% agarose gel (BIOWEST) with goldview (0.5 lg/ mL) for 30 min at 120 V in TAE buffer (40 mM Trisacetate, 1 mM EDTA, pH 8.0). The reverse primer (Chicken-R, Cattle-R, Pig-R, Horse-R) contained a species-special reverse primer (Chicken, Cattle, Pig, Horse) of at the 30 -end (in blue) and the common adapter at the 5-end (in red), respectively. The sequence of common primer (CP) (in red) was the same as the designed common adapter. The amplication fragments with only the common primer (CP) were marked in red. The amplication fragments with Common-F and the reverse primers were marked in blue.

2 For interpretation of color in Fig. 1, the reader is referred to the web version of this article.

Fig. 1. Schematic representation of Novel common primer multiplex PCR method

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3. Results 3.1. Meat identication by the reverse primers and common primers In order to compare the primer specicity of conventional PCR with that of the novel common primer PCR series of PCR assay was carried out. The qualitative 30 lL PCR reactions were run with 100 ng of genomic DNA and 15 pmol primers, respectively. The optimized PCR amplication was performed with the following cycling conditions: denaturation at 95 C for 30 s, annealing at 60 C for 30 s and extension at 72 C for 30 s for 35 cycles followed by a nal extension time of 72 C for 10 min. Fig. 2 shows 2.5% agarose gel electrophoresis of PCR products amplied from four kinds of meats. PCR products, amplied from chicken, cattle, pig and horse DNAs by species-specic reverse primers, were single DNA fragments of 221 bp, 274 bp, 394 bp and 433 bp, respectively (Fig. 2, Lanes 25). PCR products, amplied from chicken, cattle, pig and horse DNAs by the reverse primers (Fig. 2, Lanes 711), CF and CP, were single DNA fragments of 239 bp, 292 bp, 412 bp and 451 bp, respectively. Thus four kinds of meats could be identied based on the length of PCR products with no cross reaction. The bands (Lanes 69) amplied by species-special reverse primers, CF and CP were more of 18 bp than the bands (Lanes 25) amplied by the species-special reverse primers and the CF, which was identical with the theory. The amplied efciency of the primers with CP was no less than the primers without CP. 3.2. Identication of primers sensitivity The primers sensitivity was investigated, giving the example of beef and horse meats with 100 ng of genomic DNAs. CF and species-specic reverse primers were mixed in the ratios of 1:1, 1:0.1, 1:0.01, 1:0.001 and 1:0.0001 (the ratio 1 means 15 pmol primer/30 ml PCR solution). The PCR amplication using CF and species-specic reverse primers was the same as the above condition of Section 3.1. CF, CP and the reverse primers were mixed in the ratios of 1:1:1, 1: 1:0.1, 1: 1:0.01, 1: 1:0.001 and 1:1:0.0001 (the ratio 1 means 15 pmol primer/30 ml PCR solution). The optimized PCR amplication with CF , CP and the reverse primers was performed with the following cycling conditions: denaturation at 95 C for 30 s, annealing at 70 C for 10 s, annealing at 60 C for 30 s,and extension at 72 C for 30 s for 10 cycles; denaturation at 95 C for 30 s, annealing at 60 C for 30 s and extension at 72 C

for 30 s for 25 cycles followed by a nal extension time of 72 C for 10 min. Fig. 3 and Fig. 4 showed the PCR products amplied from cattle and horse meats. As the concentration of species-specic primers fell to 0.15 pmol (Lane 4). There were no bands of specic DNA fragment amplied. Therefore, the sensitivity of primers without CP was 1.5 pmol and the efciency of PCR amplication fell gradually when the concentration of species-specic reverse primers decreased. When CP was added into the CP-M-PCR reaction system, the bands of specic DNA fragment appeared yet until the concentration of the reverse primers were diluted to 0.015 pmol (Lane 10). As a result, CP greatly improved the sensitivity of PCR amplication. 3.3. Optimization of CP-M-PCR system Validation of quadruplex PCR condition was performed with single, double, three and four templates, respectively. DNAs extracted from four kinds of meats were mixed for use as templates in the same ratios (10 ng each). CF, CP, Chicken-R, Cattle-R, Pig-R and Horse-R primers were mixed in the ratios of 1:1:0.01:0.01:0.01:

Fig. 3. Agarose gel electrophoresis of PCR product amplied from cattle meat with CF, CP and reverse primers. Lane1 is a PCR product amplied from the reaction solution without template DNA; Lanes 26 with CF and species-specic primers; Lane 2, (1:1); Lane 3, (1:0.1); Lane 4, (1:0.01); Lane 5, (1:0.001) ; Lane 6, (1:0.0001); Lane 711 with CF, CP and species-specic primers; Lane 7,(1:1:1); Lane 8, (1:1:0.1); Lane 9, (1:1:0.01); Lane 10, (1:1:0.001); Lane 11, (1:1:0.0001); M is a molecular marker.

Fig. 2. Agarose gel electrophoresis of PCR product amplied from four kinds of meats. Lane1 is a PCR product amplied from the reaction solution without template DNA; Lane 25: bands amplied with CF and species-special reverse primers; Lane 2, chicken; Lane 3, cattle; Lane 4, pig; Lane 5, horse; Lanes 69: bands amplied with CF, CP and reverse primers; Lane 6, chicken; Lane 7, cattle; Lane 8, pig; Lane 9, horse; M is a molecular marker.

Fig. 4. Agarose gel electrophoresis of PCR product amplied from horse meat with CF, CP and reverse primers. Lane1 is a PCR product amplied from the reaction solution without template DNA; Lane 26 with CF and species-specic primers; Lane 2, (1:1); Lane 3, (1:0.1); Lane 4, (1:0.01); Lane 5, (1:0.001) ; Lane 6, (1:0.0001); Lane 711 with CF, CP and species-specic primers ;Lane 7,(1:1:1); Lane 8, (1:1:0.1); Lane 9, (1:1:0.01); Lane 10,(1:1:0.001); Lane 11, (1:1:0.0001); M is a molecular marker.

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Fig. 5. Agarose gel electrophoresis of PCR product amplied from four kinds of meats. Lane1 is a PCR product amplied from the reaction solution without template DNA; Lane 2, chicken; Lane 3, cattle; Lane 4, pig; Lane 5, horse; Lane 6, chicken and cattle; Lane 7, chicken and pig; Lane 8, chicken and horse; Lane 9, cattle and pig; Lane 10, cattle and horse; Lane 11, pig and horse; Lane 12, chicken, cattle and pig; Lane 13, chicken, cattle and horse; Lane 14, chicken, pig and horse; Lane 15, cattle, pig and horse; Lane 16, chicken, cattle, pig and horse; M is a molecular marker.

0.01 (the ratio 1 means 15 pmol primer/30 ml PCR solution). The optimized PCR amplication was performed with the same as PCR amplied with CF, CP and the reverse primers of Section 3.2. Fig. 5 showed 2.5% agarose gel electrophoresis of multiplex PCR products amplied from four meats. Lanes 25 were the bands of the single templates, respectively. Lanes 6-11 were the bands of the double templates, respectively. Lanes 1215 were the bands of the three templates, respectively. Lane 16 was the bands of the four templates. CP-M-PCR method that could identify the four kinds of meats had the advantage of general multiplex PCR. These primers were mixed in the simpler ratio than former conventional multiplex PCR. CP-M-PCR method had very highly amplication efciency and specicity. In addition, CP-M-PCR method conquered

Fig. 7. Agarose gel electrophoresis of PCR products amplied from 0.001, 0.01, 0.1, 1 and 10 ng DNA of the four meats. Lane 1 is a PCR product amplied from the reaction solution without template DNA. Lanes 2, 3, 4, 5 and 6 are 10, 1, 0.1, 0.01 and 0.001 ng of four meats DNA, respectively. M is DNA ladder 100 bp marker.

the failure that conventional multiplex PCR produced the lower amplication efciency because of the competition between primers and templates. 3.4. Detection limits of DNA samples In order to identify the CP-M-PCR detection limits of single DNA template, DNAs extracted from chicken, cattle, pig and horse meats were mixed for use as templates in the ratios of 1:0.0001:1:1, 1:0.001:0.1:1, 1:0.01:0.01:1, 1:0.1:0.001:1, 1:1:0.0001:1 (the ratio 1 means 100 ng template /30 ll PCR solution). CP-M-PCR amplication condition was the same as that in Section 3.3. Fig. 6 showed the bands of cattle and pig fragments of 274 and 394 bp, along with the relationships between template DNA amounts and band intensity. When cattle DNA increased, from Lane 2 to Lane 6, the bands of 274 bp fragment became more and more intense and the bands of 394 bp fragment became more and more faint. Therefore, the detection limits was 0.1 ng for single DNA templates. Subsequently, the CP-M-PCR detection limits of four DNA templates were investigated. Fig. 7 showed the results of PCR amplication from mixed DNA templates of 10, 1, 0.1, 0.01 and 0.001 ng each. Lanes 24 show four bands corresponding to the four species, thus the CP-M-PCR detection limits were 0.1 ng for all meat species. 4. Discussion The aim of this study was to develop a simple and sensitive novel PCR method for simultaneous identication of multiple meat species. CP-M-PCR method was a promising technique for this purpose. The design of primers was very important on multiplex PCR techniques, because primer specicity and melting temperature (Tm) were more critical than conventional PCR. Comparing genomic DNA sequences of chicken, cattle, pig and horse with the common adapter sequence using DNAMAN, the common adapter sequence were not matched with genomic DNA sequences. Ratios of mismatching in this study were more than 75% between the reverse primers and CF. The mismatches more than 75% made a reverse primer with a common adapter anneal only to the species-specic DNA sequence in CP-M-PCR. After a series of optimization, CP-M-PCR amplication with CF, CP and

Fig. 6. Agarose gel electrophoresis of PCR product amplied from four kinds of meats. Lane1 is a PCR product amplied from the reaction solution without template DNA.; Lane 2, chicken(100 ng), cattle(0.01 ng), pig(100 ng), horse(100 ng); Lane 3, chicken(100 ng), cattle(0.1 ng), pig(10 ng), horse(100 ng); Lane 4, chicken(100 ng), cattle(1 ng), pig(1 ng), horse(100 ng); Lane 5, chicken(100 ng), cattle(10 ng), pig(0.1 ng), horse(100 ng); Lane 6, chicken(100 ng), cattle(100 ng), pig(0.01 ng), horse(100 ng); M is a molecular marker.

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reverse primers was performed with the appropriate cycling conditions. Initially, reverse primers and CF took action for amplication of DNA samples whereas the common primer (CP), the same as sequence of the common adapter, did not almost have the target templates in the former several cycles. With the reverse primers using up and the amplied products increasing, common primer (CP) and CF was given full play to amplify the fragments of four different lengths. Primer specicity in the entire DNA of a target species was examined by single PCR using both two primers mixture (CF and species-specic reverse primers) and three primers mixture (CF, CP and reverse primers), respectively. The size of PCR products was as expected with no additional fragment from a target species. This result showed that the primers with CP amplied only one size fragment from a target species (Fig. 2). In addition, primer specicity to the other species was examined by CP-M-PCR using the same primer mixture in the method (Fig. 5). CP-M-PCR resulted in a special band of target size from four meat species and no fragment produced by non-specic amplication. In the CP-M-PCR, the common primer (CP) greatly improved the amplied sensitivity. As the CP was added into the reaction system, the band of specic DNA fragment appeared yet until the concentration of species-specic primers diluted to 0.015 pmol, which made the primer specicity enhance no less 100 times than that of conventional multiplex PCR (Fig. 3 and Fig. 4). The primers were designed to amplify target sequences of the four species at similar efciency using two primers mixture (CF and species-specic reverse primers). However, conventional multiplex PCR using equal amount mixture of the primers did not result in equal signals from the four Species (Matsunaga et al., 1999). Amplication efciency was affected by the difference of primer sequences. Because the CF primer was designed to be shared by the four species, amplication efciency of PCR was affected by only species-special reverse primers. In order to control the efciency, a common adapter was designed on species-specic reverse primers. A common primer (CP) could be effective on the identical amplied efciency. Primer sensitivity was important to the efciency of PCR amplication. In the CP-M-PCR, the common primer (CP) greatly improved the amplication sensitivity and simplied the reaction system. The new designed common primer (CP) of CP-M-PCR method had a higher 100 times sensitivity than no common primer (Fig. 3 and Fig. 4). There was the identical signal intensity using the same ratio of species-special primer mixture (Fig. 5). Owing to the application of the common adapter and the common primer, the novel CP-M-PCR method was quite useful for routine analysis of meat species identication, being quicker and more sensitive (0.1 ng) (Fig. 6 and Fig. 7). On the basis of the genomic DNA of 1.25, 3.60, 3.00, 3.15 pg per haploid genome of chicken, cattle, pig and horse, respectively (http://www.genomesize.com/), this means the sensitivity of the novel CP-M-PCR corresponds to an average of 36.4 copies of four kinds of genomic DNA in the templates when the amount of the DNA template was lowered to 0.1 ng of four kinds of meat genomic DNA. By the present method, four kinds of meats could be identied at the same time more easily and sensitive than conventional methods. The CP-M-PCR method could be widely applied in practical detection for simultaneous identication of other meat species and their products. Acknowledgments This study was funded by Beijing Municipal Commission of Education and Ministry of Science and Technology of China.

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