MEAT SCIENCE

Meat Science 68 (2004) 431438 www.elsevier.com/locate/meatsci

Detection of meat species using TaqMan real-time PCR assays

John J. Dooley
a

a,* ,

Kelly E. Paine b, Stephen D. Garrett a, Helen M. Brown

Department of Chemistry and Biochemistry, Campden & Chorleywood Food Research Association, Station Road, Chipping Campden, Gloucestershire, GL55 6LD, UK b Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire RG20 7NN, UK Received 16 February 2004; accepted 19 April 2004

Abstract Species-specic real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specic primers. For detection purposes, two TaqMan probes were developed; the rst was specic to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%. 2004 Elsevier Ltd. All rights reserved.
Keywords: TaqMan; Real-time PCR; Meat species; Cytochrome b gene

1. Introduction The need for reliable, sensitive methods for meat species identication and quantication encompasses many issues including the fraudulent substitution of cheaper meats in place of more expensive species, the inclusion of meat in non-meat (vegetarian) products or the use of lower amounts of meat than are declared on the product. This can have severe economic, ethical and medical repercussions. Existing methods for meat speciation use one of three analytes: lipids, proteins or DNA. Lipid analysis is really only suited to cases where there is a need to determine the species of origin of animal fats. The limit of detection is such that it is only suited to the detection of gross substitution. Protein analysis for speciation uses either electrophoresis or immunoassay. Of the two methods, immunoassay is the more user-friendly and has better specicity across a range of species. Com-

Corresponding author. Tel.: +44-1386-842203; fax: +44-1386842100. E-mail address: j.dooley@campden.co.uk (J.J. Dooley). 0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2004.04.010

mercial kits are available to determine the presence of a particular meat species in a mixture (Lumley, 1996). Heat denaturation of proteins during cooking and thermal processing can complicate detection of specic species. A recent report by Ozgen-Arun and Ugur (2000) describes the use of SDSPAGE to dierentiate beef, horse and pork meat in cooked sausages. Immunoassay antibodies that are specic to thermostable antigens are now commercially available in kits for cooked meat speciation. These kits provide for qualitative detection of species; however, dierentiation between poultry species is not possible. By contrast, DNA is a relatively stable molecule and has been successfully extracted from cooked and canned meats. A variety of DNA based techniques (slot blot hybridisation, post polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) and single strand conformational polymorphism (SSCP)) have been used to dierentiate various meat types. A comparison between ELISA, PCR-RFLP and slot blot hybridisation methods for meat species identication has been reported (Hargin, 1997; Lumley, 1998). Since the work of Kocher et al. (1989) the use of mitochondrial genes and in particular the cytochrome b

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(cytb) gene for meat species identication using DNA has been nearly universal (Chikuni, Tabata, Kosugiyama, & Monma, 1994; Matsunaga et al., 1999; Wolf, Rentsch, & Hubner, 1999). Matsunaga et al. (1999) reported the use of a multiplex PCR assay for detection of up to ve species. The ready availability of sequence data for the cytb gene from these studies proved useful for the development of new TaqMan assays. The TaqMan assay diers from conventional PCR methods in its use of uorescent dyes for detection purposes. The method relies on the 50 30 exonuclease activity of the Taq polymerase enzyme to cleave a duallabelled, DNA oligonucleotide (probe) which binds to a site within the PCR amplicon. Cleavage of the probe results in an increase of uorescence proportional to the amount of template DNA present. The use of uorescence for detection purposes eliminates the need for gel electrophoresis and permits real-time PCR. Restrictions on the PCR amplicon size (maximum 150 bp) lend this system to detection of DNA in highly processed food products where the likelihood of DNA degradation is high. TaqMan PCR has been used for authentication of food products including wheat species in pasta (Wiseman, 1999) and for the determination of genetically modied (GM) soya and maize in raw and processed materials (Vatilingom, Pijnenburg, Gendre, & Brignon, 1999). The aim of this study was to develop real-time PCR (TaqMan) assays which would enable sensitive detection of a range of economically important meat species, namely beef (Bos taurus), chicken (Gallus gallus), lamb (Ovis aries), pork (Sus scrofa) and turkey (Maleagris gallopavo). The development of assays for the detection of these ve species is reported.

The quencher moiety used on both probes was TAMRA. Initially assay specicity was conrmed by performing hot-start, end-point PCR with AmpliTaq Gold DNA polymerase, 300nM of each primer, 50 ng of template DNA and 5mM MgCl2 . Amplication proles (95 C [10 min] [enzyme activation] followed by 30 cycles of a two-step PCR; 95 C [15 s], 60 C [60 s]) were applied using a PE9600 PCR machine (Applied Biosystems, Warrington, UK). Product amplication was conrmed using standard agarose gel techniques (Sambrook, Fritsch, & Maniatis, 1989). Selected primers and probes were optimised for use on the Applied Biosystem 7700 PCR machine (TaqMan). Optimal primer and probe concentrations are shown in Table 1. 2.2. DNA extraction DNA was extracted from 2 g of lean meat tissue using a CTAB extraction method. Minced meat (2 g) was mixed with 10 ml CTAB-lysis buer, pH 8.0 (2% CTAB, 1.4 M NaCl, 0.1 M TrisHCl, 20 mM EDTA) in a 25 ml sterile pot and incubated at 65 C with agitation for 1 hour. Proteinase K (10 ll of a 20 mg/ml solution) was added and the sample incubated at 65 C, with agitation overnight. An aliquot (1 ml) was transferred to a 1.5 ml Eppendorf and centrifuged for 10 min at 13,000g before 800 ll of the supernatant was transferred to a fresh 1.5 ml Eppendorf. Chloroform (600 ll) was added and the sample vortexed. Following centrifugation at 13,000g for 10 min, 600 ll of the aqueous layer was removed and mixed with 500 ll of absolute isopropanol in a fresh 1.5 ml Eppendorf. After 30 min at room temperature, the DNA was recovered, cleaned and redissolved in 50 ll of sterile distilled water (SDW) (Sambrook et al., 1989). Recovered DNA was quantied at 260:280nm using a GeneQuant pro (Pharmacia). 2.3. Admixture preparation

2. Methods 2.1. Development of TaqMan assays Gene sequences for the cytochrome b (cytb) gene from the ve species of interest were obtained from GenBank. Accession numbers for the gene sequences are D34635 (beef), L08376 (chicken), X56284 (lamb), X56295 (pork) and L08381 (turkey). Sequence alignment was performed using the PileUp sequence alignment package from the GCG software suite (Wisconsin Package). Areas of consensus sequence were targeted for probe development, while regions of sequence variability were selected for species-specic primer design. Primers and probes were designed using Primer Express (Applied Biosystems, Warrington, UK). Primers and probes were obtained from Applied Biosystems. Probes were labelled with reporter dyes 6-carboxyuorescein (FAM) (mammalian probe) or 6-carboxy4,7,20 ,70 -tetrachlorouorescein (TET) (poultry probe). DNA admixtures were prepared by mixing appropriate volumes of 50 ng/ll DNA solutions from each species. A total of 500 ll of each DNA admixture was prepared and stored at )20 C in 100 ll aliquots. Meat admixtures (200 g total) were prepared by combining appropriate quantities of minced, lean meat obtained from beef (topside), pork and lamb (shoulder) or turkey and chicken (breast). DNA was extracted from meat admixtures using the CTAB method. 2.4. Assay application Optimised cytb TaqMan assays for beef, chicken, lamb, pork and turkey were applied to a total of 50 ng of DNA. Each assay was tested against DNA from all ve species, i.e. against its target species and the four remaining species to conrm assay specicity. Assays were

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Optimal concentration (nM)

also applied to DNA admixtures of one species in another and to DNA extracts from meat admixtures of one species in another. Blind meat admixtures of one species spiked (0.5%) into another (99.5%) were produced. DNA was extracted and the assays applied to determine the meat species present.

Amplicon size (bp)

116

133

149

133 200

Probe

150

175

175

150

86

3. Results 3.1. Assay development

Primer 300 900 300 300 300 900 300 300 300 300

Initial DNA sequence alignment revealed no consensus sequence between all ve species; however, the major sequence variations observed were between the poultry and mammalian gene sequences. These observations indicated that it would not be possible to design a single, universal probe around which assays for all ve species could be developed. The sequence data was, therefore, realigned as two groups comprising (i) the mammalian species and (ii) the poultry species. Realigned data revealed regions of homology around which probes could be designed. Areas of variation offered the potential of designing species-specic primers. A single probe for each group (mammal or poultry) was identied. Several primer sets for each species (up to 6 sets for chicken and pork) were also designed. Following initial screening of the primers, using end-point PCR, those primers that performed well, i.e. showed no primer-dimerisation or cross-reactivity with DNA from other species, were selected for further investigation on the TaqMan. The best primer and probes sets following TaqMan optimisation are shown in Table 1. Table 1 also shows the optimal conditions for each assay. An optimal amount of DNA, in the assay, was found to be 50 ng. This produced threshold cycle (CT ) values of between 17 and 18 for all species except chicken where CT s of 2021 were observed (Table 2). The CT value refers to the PCR cycle at which uorescence is rst detected above the background uorescence. DNA concentrations below 1ng were found to produce CT values which were too high (over 30) while CT values obtained with higher DNA concentrations indicated that PCR inhibition was occurring as evidenced by a loss of linearity between CT value and DNA concentration. This inhibition was particularly noticeable with beef DNA. Results for the chicken assay were representative of all assays and are shown in Fig. 1(a). The eects of inhibition of increased amounts of beef DNA are shown in Fig. 1(b), which clearly shows a non-linear relationship between CT and amount of DNA. These gures show that the chicken assay worked well over a wide range of starting template DNA quantities (0.1 200 ng); however, the beef assay shows results typical of PCR inhibition when more than 200 ng of template

59.8 58.6

56.3 56.7

58.9 58.4

59.4 58.3

56.9 59.3

>68 TGA GGA CAA ATA TCA TCA TTC TGA GGA GCW ARG TYA

65.8 Poultry
a

Tm

Table 1 Primers and probes for cytochrome b (cytb) single species assays

Mammala

Chicken

Turkey

Species

Lamb

Pork

Beef

FAM, 6-carboxyuorescein; TET, 6-carboxy-4,7,2 ,7 -tetrachlorouorescein; Tm , melting temperature; bp, base-pairs. Some mismatching at 30 -end was required to produce probe suitable for use with all three species.

ATG AAA CAT TGG AGT AGT CCT ACT ATT TAC C CTA CGA GGT CTG TTC CGA TAT AAG G

AGC AAT TCC CTA CAT TGG ACA CA GAT GAT AGT AAT ACC TGC GAT TGC A

ACA ACC CAA CCC TTA CCC GAT TCT TC Probe TET

GAG TAA TCC TCC TAT TTT GCG ACA AGG TTT GTG CCA ATA TAT GGA ATT

CGG AGT AAT CCT TCT GCT CAC AGT GGA TTG CTG ATA AGA GGT TGG TG

ACC CTA GTA GAG TGA GCC TGA GG AAG GGC AGG AGG AAG TGG AG

Sequence (50 30 )

Reporter moiety

Optimal primer sets

Forward 2 Reverse 2

Forward 1 Reverse 3

Forward Reverse

Forward Reverse

Forward Reverse

Probe

FAM

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Table 2 Specicity and sensitivity of cytb TaqMan assays Assay Beef Tested against Beef Chicken Lamb Pork Turkey Chicken Beef Lamb Pork Turkey Lamb Beef Chicken Pork Turkey Pork Beef Chicken Lamb Turkey Turkey Beef Chicken Lamb Pork CT a 17 40 31.5 39 37 20.34 33.27 33.37 30.05 31.87 17 37 40 37 39 17.41 31.13 37.08 30 34.65 18 40 34.5 38 40 Additional cyclesb n/a 23 14.5 22 20 n/a 12.93 13.03 9.71 11.53 n/a 20 23 20 22 n/a 13.72 19.67 12.59 17.24 n/a 22 16.5 20 22 % Cross-reactivity 100.0 0.000 0.004 0.000 0.000 100.0 0.010 0.008 0.098 0.024 100.0 0.000 0.000 0.000 0.000 100.0 0.009 0.000 0.018 0.001 100.0 0.000 0.001 0.000 0.000 Theoretical limit of detection at CT 30 (%) 0.012

Chicken

0.1

Lamb

0.01

Pork

0.02

Turkey

0.02

a b

CT obtained using 50 ng of DNA. Note that if PCR is restricted to 30 cycles no cross-reactivity is detected. CT for species tested against minus CT for assay specic (target) species.

DNA was used. Subsequent work was, therefore, performed using a total of 50 ng of template DNA in each assay. Amplicon size restrictions imposed during the development of TaqMan assays requires that targets of no more than 150 bp are produced. These small amplicons are ideal for use with processed foods where DNA degradation can mean that larger targets (>200 bp) are not always amplied. Preliminary work performed by us (not reported here) has shown that the assays developed here can be used to detect meat species in admixtures following processing at temperatures of 121 C for 15 min. Application of the standard 40 cycle TaqMan PCR reaction resulted in some cross-reactivity between some assays and DNA of other non-target species. A dierence of 1 CT value between the target species and the non-target species represents a cross-reactivity of 50% as, in theory, double the amount of non-target species DNA would be required to give the same CT as the target species. The percentage cross-reactivity is thus halved for each extra CT . Results showed little (less than 0.005%) or no cross-reactivity for the beef, lamb and turkey assays after 40 cycles (Table 2). Higher crossreactivity was observed with the pork and chicken

assays. Pork, lamb, beef and turkey DNA produced cross-reactivities between 0.01% and 0.1% with the chicken assay while beef and lamb yielded crossreactivities of 0.009% and 0.018%, respectively, with the pork assay. It was concluded that the level of crossreactivity, even in the chicken assay, would not lead to errors in species identication if a CT of 30 was used as the cut-o point for positive identication of a species in raw meat admixtures. 3.2. Limit of detection The theoretical limit of detection for each assay, using a cut-o CT of 30, was determined from crossreactivity levels obtained previously. These are shown in the last column of Table 2. To determine the limits, the amount of target (specic species) and non-target (non-specic species) species DNA required to obtain a CT of 30 was calculated. The proportion of target DNA in non-target DNA was determined as a percentage which equates to the limit of detection. All values obtained for the theoretical levels were below or equal to 0.1%. Assays for beef, lamb and turkey were applied to DNA admixtures (0.5% and 0.1%) of one species in

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Chicken assay applied to chicken DNA

30 25

Ct values

20 15 10 5 0 0.01 0.1
y = -1.478Ln(x)+ 22.972 R = 0.9978
2

(a)
25 20

1 10 Amount of template DNA (ng)

100

1000

Beef assay applied to beef DNA

Ct values

15
y = -1.4266Ln(x) + 21.304

y = -0.90Ln(x)+20.40 R = 0.71
2

10 5 0 0.1 1

R = 0.9687

(b)

10 Amount of template DNA (ng)

100

1000

Fig. 1. Relationship between the amount of template DNA in the assay and the CT values for each species. (Amounts of DNA ranged from 0.1 to 500 ng). A good linear relationship between CT and DNA concentration was obtained when the chicken assay was applied to chicken DNA (a). The eects of PCR inhibition can be seen when greater than 100 ng of beef DNA was used as the template for the beef assay (b).

another to determine the experimental lower limits of detection. Using 30 cycles of PCR results showed that when spiked at 0.5%, the species could easily be detected with their respective assay (Table 3). From Table 3 it can be seen that observed CT s for each assay are about eight

cycles higher than those observed when the assays were applied to pure meats. This is as expected since halving the amount of template DNA increases the CT by 1. In this case the dierence between 50 ng (pure meat) of DNA and 0.25 ng (0.5% spike) is about 28 , which is

Table 3 Limits of detection (CT < 30) for single-species assays using DNA admixtures Assay target species Beef Spike species Mixer species CT for single species DNA (50 ng) 16 n/t n/t n/t n/t 17 36 40 37 39 18 40 34 40 40 Level of detection of spike DNAa observed CT s for spike in mixer 0.50% Detection 0.10% Detection

Beef Chicken Lamb Pork Turkey

24 24 24 24 27 26 26 26 27 26 26 26

+ + + + + + + + + + + +

n/t 27 n/t n/t 29 n/t 28 n/t n/t 28 n/t n/t

n/t + n/t n/t + n/t + n/t n/t + n/t n/t

Lamb

Lamb Beef Chicken Pork Turkey

Turkey

Turkey Beef Chicken Lamb Pork

Detection (+) of a species was dened by a CT of less than 30 being observed. Some assays were not tested (n/t).

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Table 4 Summary of results from the blind analysis of a range of raw meat admixtures Total assays performed Total number correct % correct assays 32 26 81 Correct identication Times used 0.5% Spikes (29 assays) Beef Chicken Lamb Pork Turkey 99.5% Meat mixer (29 assays) Beef Chicken Lamb Pork Turkey 5 4 7 9 4 5 7 5 5 7 Number 1 4 6 9 3 4 7 5 5 7 % 20 100 85 100 75 80 100 100 100 100

The ve single species assays were applied to 0.5% meat admixtures.

about eight cycles of PCR. The three assays were also used satisfactorily to detect their respective DNA when spiked at 0.1%. Observed CT values (shown in Table 3) ranged between CT 27 (beef in lamb) and CT 29 (lamb in beef), which is close to the 30 cycle cut-o limit. This indicates that these assays are at their practical limits of detection at 0.1%. 3.3. Application of cytb TaqMan assays to detect 0.5% of target species in raw meat admixtures The cytb assays were used to test raw meat admixtures at 0.5% spike levels to determine the misclassication rate of meat species identied at this level. All analyses were carried out on DNA prepared from blind admixtures. The ve species-specic assays were performed in duplicate on a single plate. Samples were deemed to be correctly classied when only the two meats in the admixture were identied as present. The presence or absence of a species in a sample was dened using a cut-o CT equal to 30. A species was considered present if a CT of less than 30 was obtained with the respective assay. It was noted that the CT values obtained from these assays gave a relative indication of the percentage meat content in the admixture, i.e. low CT s (around 19) were indicative of a high percentage meat species content where as CT s closer to 30 indicated a low level of meat species in the admixture. Data from 32 samples are summarised in Table 4, which shows overall assay performance and individual assay performance against both the spike or the main meat species. Table 4 also shows species identication when used as either the spike or the meat mixer in admixtures. In this case if a meat was correctly determined

as present, regardless of the other assay, it was scored as correct. From 32 samples, 26 were correctly determined as containing the species used in preparation of the admixture. Of the 6 other samples; beef could not be detected in 4 samples made with 0.5% beef; pork and lamb were identied in a sample made with turkey and lamb; and lamb was not detected in a sample containing 0.5% lamb in beef. These six specic samples were repeated on a separate occasion and all, except the 0.5% lamb in beef, produced correct results (data not shown).

4. Discussion Assays for the ve species were developed around two probes. This was necessary to overcome major sequence variations in the cytb gene, which occurred during the evolutionary divergence of the two classes of vertebrate, birds (avis) and mammals (mammalia). The use of two probes had the added advantage of conferring an extra level of specicity to the assays, which meant crossreactivity between mammalian assays and the avian species, and poultry assays and the mammalian species was minimal. The development of ve individual species-specic probes, one for each assay, could increase this specicity further and would also allow the development of the assays at regions of the sequence which show the greatest inter-species variation. Although not investigated here, the use of two probes also oers the potential to develop multiplex assays for the simultaneous detection of a mammalian species with an avian species, or vice-versa, in a single reaction. Assay specicity was achieved using species-specic primer sets, which were developed from a larger number

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of potential primer sets. Primers were tested for specicity using end-point PCR under TaqMan conditions, i.e. 5mM MgCl2 . Pre-screening of primers in this way helped to ensure that full TaqMan assays were likely to be highly specic, which reduced the need to redesign assays at this stage. This was benecial not only for reducing labour but also in eliminating the production of expensive probes, which would be of no further use for species detection. During primer testing, several primers sets were found to show cross-reactivity with other species, including species from the other class, e.g. some chicken primers amplied pork DNA. Although cross-reactivity of the latter type (poultry to mammal or vice-versa) is unlikely to be detected in the complete TaqMan assay, due to increased specicity introduced by the probe, it is undesirable to amplify extraneous PCR products if accurate quantitative analysis (the ultimate aim of this work) is to be performed. Primers were, therefore, only selected if they exhibited no crossreactivity with any other species or, in the case of the chicken and pork primers, where this was minimal. Of those primers which did exhibit cross-reactivity, a large proportion revealed cross-reactivity between pork and chicken, which was unexpected. From sequence alignments pork appeared to be less related to beef and lamb than they were to each other, which may explain why there was little cross-reactivity between pork and these two species; however, from the data it was not possible to determine why cross-reactivity between pork and chicken occurred. The small amplicon size (<150 bp) used in these assays is a requirement of the development of TaqMan type real-time assays; however, the small target size is also advantageous for detection of material in processed foods, such as cooked meat products, where extensive DNA degradation has been observed. Preliminary work, using single meats only, suggests that these assays could be employed for detection of target species in cooked products. Investigations into the use of these assays for the detection of meat species in cooked meat admixtures is ongoing. Assays developed during this work were, theoretically, able to detect 0.1% of their respective target species. This was demonstrated using DNA admixtures (Table 3); however, for the purposes of this study it was only necessary to show that detection in meat admixtures at 0.5% (or 5 g= kg) was possible. Detection at this level was readily achieved for all assays in all meat admixtures except when beef was the main component, in which case there was evidence of PCR inhibition. Inhibition of the beef assay had been noted when beef DNA was used as a template in PCRs at greater than100 ng; however, 50 ng of template DNA did not appear to cause inhibition of this assay Fig. 1b. PCR inhibition of each assay by DNA from the other species was not tested in this study; however, subsequent investigations

have shown that beef DNA, even 50 ng, can cause PCR inhibition of some assays. Work performed elsewhere (H. Hird, pers. commun.) has also shown that the CTAB extraction method, which was used here, may contribute to PCR inhibition. It is likely, therefore, that detection levels reported here could be improved if alternative DNA extraction methods, which overcome problems associated with PCR inhibition, were developed. The misclassication of some of the 32 samples analysed in this study may have been caused by the effects of using too much template DNA (i.e. PCR inhibition) even though results obtained during assay development suggested that this should not have occurred when 50 ng of template DNA was used. PCR inhibition can be overcome by using less template DNA; however, this leads to an increase in CT values and a lack of detection sensitivity due to reduced amounts of target. The analysis of a sample using several PCRs with dierent amounts of template DNA to overcome possible inhibitory eects would provide a possible solution to the problem of inhibition. Until the causes of PCR inhibition are fully understood this should be advised, especially when DNA extracts are suspected to contain beef DNA. The aim of this work was to develop new DNA detection assays that would enable sensitive identication of ve common meat species in raw and cooked meat products. This study has shown that it is possible to develop such specic assays based on real-time (TaqMan) PCR techniques. The use of 30 cycles of PCR (CT 30) ensured that assays were reliable yet sensitive enough to detect their respective target meat species, even when it formed as little as 0.5% of a raw meat admixture. It is also possible that the primers developed in this study could be used in end-point PCR for the detection of the meat species; however, the use of realtime PCR oers the ability to quantify levels of target sequence within the DNA extract. Investigations into the use of real-time (TaqMan) assays for quantitative meat analysis are on-going. The assays described here are a signicant step forward in the development of reliable, accurate qualitative assays for meat in meat products. It should also be possible to develop additional real-time assays for the detection of a wider range of species (including mammal, poultry and sh) used by the food industry.

Acknowledgements Thanks are oered to Sabine Quitard and Alice Banks for technical assistance during this work and to Kevin Hargin and Applied Biosystems for advice and support. This work was funded by The Food Standards Agency.

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J.J. Dooley et al. / Meat Science 68 (2004) 431438 Ozgen-Arun, O., & Ugur, M. (2000). Animal species determination in sausages using an SDSPAGE technique. Archiv fur Lebensmittelhygiene, 51, 4953. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning (2nd ed.). New York, USA: Cold Spring Harbour Laboratory Press. Vatilingom, M., Pijnenburg, H., Gendre, F., & Brignon, P. (1999). Real-time quantitative PCR detection of genetically modied maximizer maize and roundup ready soybean in some representative foods. Journal of Agricultural and Food Chemistry, 47, 5261 5266. Wisconsin Package Version, Genetics Computer Group (GCG), Madison, WI. Wiseman, G. (1999). Quantitative PCR detection of T. aestivum adulteration in commercial T. durum pasta using PSR 128 primers: Optimisation. Final Report for MAFF project ANO667. Wolf, C., Rentsch, J., & Hubner, P. (1999). PCR-RFLP analysis of mitochondrial dna: a reliable method for species identication. Journal of Agricultural and Food Chemistry, 47, 1350 1355.

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