Meat Science 85 (2010) 531536

Contents lists available at ScienceDirect

Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay
Snia Soares a,b, Joana S. Amaral a,b, Isabel Mafra a,*, M. Beatriz P.P. Oliveira a
a b

REQUIMTE, Servio de Bromatologia, Faculdade de Farmcia, Universidade do Porto, Rua Anbal Cunha, 164, 4099-030 Porto, Portugal ESTiG, Instituto Politcnico de Bragana, Campus de Sta. Apolnia, 5301-857 Bragana, Portugal

a r t i c l e

i n f o

a b s t r a c t
A species-specic duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of pork and poultry meat species using the mitochondrial cytb and 12S rRNA as target genes for pork and poultry, respectively. By the amplication of binary reference meat mixtures, a linear normalised calibration curve was obtained using the uorescence intensities of PCR products for pork (149 bp) and poultry (183 bp) species. The proposed method allowed the quantication of pork meat addition to poultry meat in the range of 175%, with a sensitivity of 0.1%. The in-house validation using samples with known amounts of pork meat (1.0%, 2.5%, 7.5%, 20.0% and 40%) evidenced a high reproducibility of the methodology (coefcient of variation from 4.1% to 7.6%). The successful application of the duplex PCR was also demonstrated by the high correlation (R2 = 0.99) obtained from regression analysis between the predicted and the actual values of pork meat addition in blind meat mixtures. The suggested methodology presents a low cost, fast, easy and reliable alternative to estimate the level of poultry meat adulteration by the addition of pork meat. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 7 July 2009 Received in revised form 11 February 2010 Accepted 2 March 2010

Keywords: Duplex PCR Species identication Pork Poultry Meat Authenticity

1. Introduction Food composition and authenticity assessment is becoming a very important issue by avoiding unfair competition among producers, allowing consumers to have accurate information about the acquired products. Following the European Union labelling regulations, meat products should be accurately labelled regarding their species content (European Commission, 2001). In the last years, meat species adulteration in ground and comminuted products has been a widespread problem in retail markets (Asensio, Gonzlez, Garca, & Martn, 2008). Quality and authenticity evaluation in these products encompasses many issues, such as the fraudulent substitution of higher commercial value meats by lower value meats (Fajardo et al., 2008), the presence of undeclared species (Aida, Man, Wong, Raha, & Son, 2005) and the use of vegetable proteins, since they have a considerably lower price than muscle proteins (Belloque, Garca, Torre, & Marina, 2002). Furthermore, the presence of undeclared ingredients may be troublesome for health reasons, such as in the case of bovine spongiform encephalopathy due to the addition of infected neurological tissue (Sultan et al., 2004) and because of allergic reactions in sensitised individuals (Asensio et al., 2008; Belloque et al., 2002). Another problem to be considered in meat species adulteration is related to religious practices as in some religions, such as Islam and Judaism pork meat consumption is forbidden.
* Corresponding author. Tel.: +351 222078902; fax: +351 222003977. E-mail address: isabel.mafra@ff.up.pt (I. Mafra). 0309-1740/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.03.001

Several analytical techniques have been suggested for the identication of meat species in mixed samples, including different protein-based methods such as high-performance liquid chromatography, electrophoretic techniques and enzyme-linked immunosorbent assays (Aida et al., 2005; Rodrguez, Garca, Gonzlez, Hernndez, & Martn, 2005). Although the species of origin in raw meats can be identied by using most of these protein-based methods, some authors referred that they are signicantly less sensitive in the evaluation of thermally processed foods because of specic epitopes alterations (Hird, Goodier, & Hill, 2003; Rodrguez et al., 2005). Other analytical tools based on spectroscopic methods have also been used for meat authentication because they are non-destructive, fast and low cost techniques. To overcome the large amount of generated data, the combination of spectroscopy with chemometrics is essential (Arvanitoyannis & van Houwelingen-Koukaliaroglou, 2003). The application of multivariate analysis can be also a powerful tool to evaluate the quality of meat products (Arvanitoyannis, Bloukas, Pappa, & Psomiadou, 2000; Pappa, Bloukas, & Arvanitoyannis, 2000) and to predict their safety (Maratagas, Skandamis, Nychas, & Drosinos, 2007), with the ability of correlating several chemical and sensorial parameters for the identication of consumers preferences (Papadima, Arvanitoyannis, Bloukas, & Fournitzis, 1999). More recently, DNA molecules have been choose as target compounds due to their high stability compared to proteins, and to their ubiquity in every type of cell. The analysis of DNA coupled with polymerase chain reaction (PCR) presents a fast, sensitive and highly specic alternative to protein-based methods (Mafra,

532

S. Soares et al. / Meat Science 85 (2010) 531536

Ferreira, & Oliveira, 2008). Conventional PCR techniques are generally able to produce qualitative results of the identied species, while real-time PCR has demonstrated to be a useful tool for the determination of minute amounts of different species, even in complex foodstuffs (Fajardo et al., 2008; Mafra, Ferreira, et al., 2008; Mafra, Silva, Moreira, Ferreira da Silva, & Oliveira, 2008; Sawyer, Wood, Shanahan, Gout, & McDowell, 2003). Real-time PCR is based on the use of a uorescent dye or probe, which is able to give a proportional result to the initial amount of target DNA, enabled by the real-time monitoring of amplication products along each cycle (Dooley, Paine, Garrett, & Brown, 2004; Fajardo et al., 2008). Nowadays, real-time PCR is probably the most used quantitative DNA-based method. However, the high cost of the equipment and reagents is still a drawback for the application of this technique in most laboratories. Alternatively, other approaches based on PCR for quantitative analysis have been tempted in different areas (Bauer, Polzin, & Patzelt, 2003; Burgess & Davison, 1999; Mafra, Ferreira, Faria, & Oliveira, 2004; Mafra, Roxo, Ferreira, & Oliveira, 2007). In the works of Mafra et al. (2004, 2007), simple approaches based on species-specic duplex PCR and on the use of normalised calibration curves were developed to detect and quantify cow milk addition in ovine and goat cheeses. In the present work, we describe an approach based on a duplex PCR assay for the simultaneous identication of pork and poultry species and for the estimation of the adulteration of poultry meat with pork addition by the use of a normalised calculation of the uorescent intensity of PCR products. 2. Materials and methods 2.1. Samples Reference samples were prepared in the laboratory with poultry and pork muscles acquired in a local retail market. Immediately after purchase, both meats were cut and the outside portion was rejected. The samples were then minced separately and reference binary mixtures containing 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, 99%, 99.5%, and 99.9% (w/w) of pork in poultry meat were prepared to a nal weight of 100 g. After the addition of 15 mL of sterile phosphate-buffered saline (136 mM NaCl, 1.4 mM KH2PO4, 8.09 mM Na2HPO412H2O, and 2.6 mM KCl, pH 7.2), each mixture was homogenised using a blender. To avoid contaminations, each mixture was processed separately using different material and different blender containers, previously treated with DNA decontaminator solution. To validate the estimation approach, blind validation samples containing 1%, 2.5%, 7.5%, 20% and 40% of pork in poultry meat (w/w) were also prepared. The binary reference mixtures and validation meat samples were immediately stored at 20 C after preparation until DNA extraction. 2.2. DNA extraction DNA was extracted using the Wizard method as described by Mafra, Silva, et al. (2008) with minor modications. Briey, 200 mg of grounded and homogenised samples were transferred

to a 2 mL sterile reaction tube followed by the addition of 860 lL of TNE extraction buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% SDS), 100 lL of 5 M guanidine hydrochloride solution and 40 lL proteinase K solution (20 mg mL1). After the incubation at 60 C for 3 h, with occasional stirring, the suspension was centrifuged (15 min, 18,514 g) and 500 lL of the supernatant were mixed with 1 mL of Wizard DNA purication resin (Promega, Madison, WI, USA). The mixture was eluted through a column and the resin was washed with 2 mL of isopropanol solution (80%, v/v). After drying the column, the DNA was eluted by centrifugation (1 min 10,000 g) with 100 lL of TrisEDTA buffer (10 mM Tris, 1 mM EDTA) at 70 C to a new reaction tube. The extractions were performed in duplicate assays for each binary mixture. The quality of extracted DNA was analysed by electrophoresis in a 1.0% agarose gel carried out in TAE buffer (40 mM Trisacetate, 1 mM EDTA) for 40 min at 120 V, stained with ethidium bromide (0.4 lg mL1 for 5 min) and destained in distilled water for 20 min. The agarose gel was visualised under UV light and a digital image was obtained using a Kodak Digital Science DC120 (Rochester, NY, USA). 2.3. DNA quantication and purity The DNA was quantied by spectrophotometry using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan), as described by ISO 21571 (2005) and Somma (2006). The DNA concentration was determined by UV absorbance at 260 nm (1 absorbance unit corresponds to 50 lg mL1 of dsDNA). The purity of the extracted DNA was determined by the ratio of the absorbance at 260 and 280 nm. 2.4. Oligonucleotide primers The oligonucleotide primers used in this work are presented in Table 1. The primers were synthesised by Eurons MWG Operon (Ebersberg, Germany). 2.5. PCR conditions 2.5.1. Simplex PCR The amplications by PCR were carried out in 25 lL total reaction volume containing 2 lL of DNA extract. The reaction targeting pork species contained 20 ng of DNA, 15 mM TrisHCl (pH 8.3), 50 mM KCl, 0.4 lM of each primer Pork-F/Pork-R, 0.2 mM of each dNTP (Invitrogene, Carlsbad, CA, USA), 1 mM of MgCl2 and 1 U of DNA polymerase AmpliTaq Gold (Applied Biosystems, Branchburg, NJ, USA). The reaction targeting poultry species contained 50 ng of DNA, 15 mM TrisHCl (pH 8.3), 50 mM KCl, 0.5 lM of each primer Gal-F/Gal-R, 0.2 mM of each dNTP, 2.25 mM of MgCl2 and 1 U of DNA polymerase AmpliTaq Gold (Applied Biosystems, Branchburg, NJ, USA). The PCR amplications were performed in a thermal cycler PTC100 (MJ Research, Inc., Watertown, MA, USA). The following programs were used: initial denaturation at 94 C for 5 min; 35 and 38 cycles for pork and poultry species, respectively, at 94 C for 30 s, 60 C for 60 s and 72 C for 60 s; and a nal extension at

Table 1 Oligonucleotide primers. Primer Pork-F Pork-R Gal-F Gal-R Sequence (50 30 ) ATG AAA CAT TGG AGT AGT CCT ACT ATT TAC C CTA CGA GGT CTG TTC CGA TAT AAG G TGA GAA CTA CGA GCA CAA C GGG CTA TTG AGC TCA CTG TT Target cytb rRNA 12S Amplicon (bp) 149 183 Reference Dooley et al. (2004) Dalmasso et al. (2004)

S. Soares et al. / Meat Science 85 (2010) 531536

533

72 C for 5 min. The amplied fragments were analysed by electrophoresis in a 2.0% agarose gel carried out in TAE buffer (40 mM Trisacetate, 1 mM EDTA) for 60 min at 120 V, stained with ethidium bromide (0.4 lg mL1 for 5 min) and destained in distilled water for 30 min. The agarose gel was visualised under UV light and a digital image was obtained using a Kodak Digital Science DC120 (Rochester, NY, USA). 2.5.2. Duplex PCR The amplications by duplex PCR were carried out in 25 lL total reaction volume containing 2 lL of DNA extract (20 ng), 15 mM TrisHCl (pH 8.3), 50 mM KCl, 0.1 lM of each primer Pork-F/ Pork-R, 0.24 lM of each primer Gal-F/Gal-R, 0.2 mM of each dNTP, 2 mM of MgCl2 and 1 U of DNA polymerase AmpliTaq Gold (Applied Biosystems, Branchburg, NJ, USA). The PCR amplications were performed in a thermal cycler PTC-100 using the program: initial denaturation at 94 C for 5 min; 2535 cycles at 94 C for 30 s, 60 C for 60 s and 72 C for 60 s; and a nal extension at 72 C for 5 min. The amplied fragments were analysed by electrophoresis in a 3.0% agarose gel carried out in TAE buffer for 90 min at 120 V, stained with ethidium bromide (5 min) and destained in distilled water (30 min). The agarose gel was visualised under UV light and a digital image was recorded.

2.6. Statistical analysis A regression analysis between actual and estimated amounts of pork meat with 95% condence interval was performed. SPSS for Windows version 17.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical treatment of results. 3. Results and discussion 3.1. Qualitative and quantitative analysis of extracted DNA The quality of DNA extracts from reference binary meat mixtures was examined by agarose gel electrophoresis (data not shown). This enabled to verify the DNA integrity since variations of DNA fragment length, which is a parameter for DNA integrity, are dependent on the material under examination, the degree of processing and the DNA extraction method. In general, the binary mixtures showed intense smears of short and long DNA molecules (>10,000 bp), indicating that DNA was not excessively degraded during the Wizard extraction protocol, as previously reported for other DNA extracts (Mafra, Silva, et al., 2008). The purity of the extracts obtained from all the standard binary meat mixtures was high, since the ratio A260/A280 ranged be-

A
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C

149 pb

B
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C

183 bp

Fig. 1. Agarose gel electrophoresis of PCR products targeting pork (A) and poultry species (B) in binary reference mixtures of pork/poultry meats. M: 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1: 100% poultry; lanes 215: 0.1%, 0.5%, 1%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, 97.5%, 99%, 99.5% and 99.9% (w/w) of pork addition, respectively; lane 16: 100% pork; C: negative control.

534

S. Soares et al. / Meat Science 85 (2010) 531536

tween 1.7 and 1.8. The DNA yields ranged between 716 and 1034 ng/lL. The high purity of extracts and high DNA yields suggest the adequacy of the Wizard extraction protocol for meat based products. 3.2. Simplex PCR In a preliminary phase of this work, simplex PCR assays were carried out for individual species identication in meat mixtures and to evaluate the sensitivity of each reaction. As expected, the amplication of DNA extracts of binary reference mixtures yielded the PCR fragments of 149 and 183 bp for pork and poultry species, respectively (Fig. 1). The results showed the applicability of the primers proposed by Dooley et al. (2004) for the identication of pork species and by Dalmasso et al. (2004) for the identication of poultry species, both in meat products. The sensitivity of PCR targeting pork DNA was veried until the minimum amount tested of 0.1% of pork addition to poultry meat (Fig. 1A). The same sensitivity was observed for the specic detection of poultry meat (Fig. 1B). 3.3. Duplex PCR The same primers used in simplex PCR were applied in duplex PCR amplication. After optimisation of the technique using the reference binary meat mixtures, it was possible to obtain the detection of 0.1% of pork species in poultry meat by the simultaneous amplication of both targets, namely, the mitochondrial cytb for pork and the mitochondrial 12S rRNA for poultry (Fig. 2). Although the sensitivity of multiplex PCR amplications is known to decrease comparing with the one target reaction, by using the proposed duplex PCR it was possible to reach the same level of detection as for the simplex PCR for pork species (0.1%). This result evidences that the developed duplex PCR can easily be used for screening poultry meat adulteration with pork addition with high sensitivity. Other authors have also reported the use of duplex or multiplex PCR techniques for meat species identication. Di Pinto, Forte, Conversano, and Tantillo (2005) used a duplex PCR for the detection of porks meat in fresh sausages prepared with horses meat (no levels of detection reported). Dalmasso et al. (2004) applied a multiplex PCR for the simultaneous detection of ruminant, poultry, sh and pork material in feedstuffs, reporting a detection

limit of 0.002% for pork species. The duplex PCR proposed by Krcmar and Rencova (2003) allowed the detection of 0.01% of swine and chicken meat-and-bone meal. Matsunaga et al. (1999) reported a multiplex PCR assay for the identication of cattle, pig, chicken, sheep, goat and horse meat, with a detection limit of 0.25 ng DNA for all species. The duplex PCR assay was then optimised for a semi-quantitative approach analysing the PCR band intensities after agarose gel electrophoresis, as described by Mafra et al. (2004, 2007) for species identication in cheeses. Using image analysis software, the

A 0.8
0.6

Npork

0.4 0.2 0

0 0 -0.1

20

40

60

80

% pork

-0.5

0.5

1.5

log Npork

-0.2 -0.3 -0.4

y = 0.0954x - 0.3464 R2 = 0.9959

log (%pork)

Fig. 3. Normalised calibration curves for the estimation of poultry meat adulteration with pork obtained with a 35-cycle duplex PCR. Npork is the normalised band intensity for pork species calculated with image analysis software Kodak Digital Science. (A) Relative band intensities vs. pork meat percentage; (B) logarithm of relative band intensities vs. logarithm of pork meat percentage. Values are mean of replicate assays (n = 2).

10

183 bp 149 bp

Fig. 2. Agarose gel electrophoresis of duplex PCR products from binary reference mixtures of pork/poultry meats amplied with 35 cycles. M: 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1: 100% poultry; lanes 29: 0.1%, 0.5%, 1%, 5%, 10%, 25%, 50% and 75% (w/w) of pork addition, respectively; lane 10: 100% pork; C: negative control.

S. Soares et al. / Meat Science 85 (2010) 531536

535

10

11

12

13

183 bp 149 bp

Fig. 4. Agarose gel electrophoresis of duplex PCR products from binary reference mixtures of pork/poultry meats amplied with 25 cycles. M: 100 bp ladder (Bioron, Ludwigshafen, Germany); lane 1: 100% poultry; lanes 27: 1%, 5%, 10%, 25%, 50% and 75% of pork addition, respectively; lane 8: 100% pork; lanes 913: 1%, 2.5%, 7.5%, 20% and 40% (w/w) of pork addition (blind validation samples), respectively; C: negative control.

uorescence intensities of the PCR bands obtained by the simultaneous amplication of pork and poultry DNA from the reference binary meat mixtures were correlated with the pork meat percentage. To overcome variations that might occur during DNA extraction, amplication and gel preparation, the intensity of the target band was normalised. For that purpose, considering that in optimal conditions the sum of the two bands (pork + poultry) should be constant, the target band intensity can be related to the sum of the intensities of the two bands. Thus, the normalised band intensities for pork species were calculated using the following expression:

Npork

Ipork Ipork Ipoultry

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

where Npork is the normalised band intensity for pork, Ipork and Ipoulare the band intensities for pork and poultry, respectively. Fig. 3A shows the Npork vs. the percentage of pork meat in the reference binary meat mixtures. Fig. 3B depicts the same relationship, but using a logarithmic scale in both axes, which enables a linear normalised calibration curve with a high correlation coefcient in the range of 0.175%. To increase the response factor (given by the slope of the equation), the number of PCR cycles was optimised to decrease band saturation, without excessively compromising the sensitivity of the method. A PCR of 25 cycles (Fig. 4, lanes 18) was considered a good compromise among linearity, response factor of the method (0.449), and sensitivity (1%) (Fig. 5), which is in good agreement with the results obtained for the duplex PCR applied to bovine species quantication in dairy products (Mafra et al., 2004).
try

N pork

3.4. Prediction of pork meat addition The prediction of the amount of pork meat in blind samples was carried out using the standard curve in the range of 175% obtained with a 25-cycle duplex PCR (Fig. 4, lanes 913, and Fig. 5). Table 2 shows the predicted and actual values of those samples for the validation of the proposed quantitative model. The results show that the predicted values of pork meat addition were close to the actual percentage values. The low variability among repli-

20

40

60

80

% pork

0.0 0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2

0.5

1.0

1.5

2.0

log N pork

y = 0.4452x - 0.9487 R 2 = 0.9891

Table 2 Determination of pork meat percentage in validation samples by a duplex PCR using a calibration curve with 25 cycles (Fig. 5B). Samples Pork meat (%) Actual Mean predicted 1.32 2.68 11.6 22.9 38.9
a

SDb

CVc (%)

log (%pork)

Fig. 5. Normalised calibration curves for the estimation of poultry meat adulteration with pork obtained with a 25-cycle duplex PCR. Npork is the normalised band intensity for pork species calculated with image analysis software Kodak Digital Science. (A) Relative band intensities vs. pork meat percentage; (B) logarithm of relative band intensities vs. logarithm of pork meat percentage. Values are mean of replicate assays (n = 8). The errors bars are the standard deviations.

1 2 3 4 5
a b c

1.0 2.5 7.5 20.0 40.0

0.039 0.031 0.03 0.02 0.02

4.32 4.08 6.26 6.65 7.61

Values are the mean of replicate assays (n = 7). Standard deviation. Coefcient of variation.

536

S. Soares et al. / Meat Science 85 (2010) 531536 Arvanitoyannis, I. S., Bloukas, J. G., Pappa, I. C., & Psomiadou, E. (2000). Multivariate data analysis of cavourmas A Greek cooked meat product. Meat Science, 54, 7175. Arvanitoyannis, I. S., & van Houwelingen-Koukaliaroglou, M. (2003). Implementation of chemometrics for quality control and authentication of meat and meat products. Critical Reviews in Food Science and Nutrition, 43, 173218. Asensio, L., Gonzlez, I., Garca, T., & Martn, R. (2008). Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control, 19, 18. Bauer, M., Polzin, S., & Patzelt, D. (2003). Quantication of RNA degradation by semi-quantitative duplex and competitive RT-PCR: A possible indicator of the age of bloodstains? Forensic Science International, 138, 94103. Belloque, J., Garca, M. C., Torre, M., & Marina, M. L. (2002). Analysis of soyabean proteins in meat products: A review. Critical Reviews in Food Science and Nutrition, 42, 507532. Burgess, S. C., & Davison, T. F. (1999). A quantitative duplex PCR technique for measuring the amounts of cell-associated Mareks disease virus: Differences in two populations of lymphoma cells. Journal of Virological Methods, 82, 2737. Dalmasso, A., Fontanella, E., Piatti, B., Civera, T., Rosati, S., & Bottero, M. T. (2004). A multiplex PCR assay for the identication of animal species in feedstuffs. Molecular and Cellular Probes, 18, 8187. Di Pinto, A., Forte, V. T., Conversano, M. C., & Tantillo, G. M. (2005). Duplex polymerase chain reaction for the detection of pork meat in horse meat fresh sausages from Italian retail sources. Food Control, 16, 391394. Dooley, J. J., Paine, K. E., Garrett, S. D., & Brown, H. M. (2004). Detection of meat species using TaqMan real-time PCR assays. Meat Science, 68, 431438. European Commission (2001). Directive 2001/101/EC of 26 November 2001 amending Directive 2000/13/EC of the European Parliament and of the Council on the approximation of 20 March 2000 on the approximation of the laws of the Member States relating to the labelling, presentation and advertising of foodstuffs. Ofcial Journal of the European Communities L, 310, 1921. Fajardo, V., Gonzlez, I., Martn, I., Rojas, M., Hernndez, P. E., Garca, T., et al. (2008). Real-time PCR for detection and quantication of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Science, 79, 289298. Hird, H., Goodier, R., & Hill, M. (2003). Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisation with vistra green. Meat Science, 65, 11171123. ISO 21571 (2005). Foodstuffs Methods of analysis for the detection of genetically modied organisms and derived products Nucleic acid extraction (1st ed.). Geneva: International Standard ISO 21571, ISO. Krcmar, P., & Rencova, E. (2003). Identication of species-specic DNA in feedstuffs. Journal of Agricultural and Food Chemistry, 51, 76557658. Mafra, I., Ferreira, I. M. P. L. V. O., Faria, M. A., & Oliveira, M. B. P. P. (2004). A novel approach to the quantication of bovine milk in ovine cheeses using a duplex polymerase chain reaction method. Journal of Agricultural and Food Chemistry, 52, 49434947. Mafra, I., Ferreira, I. M. P. L. V. O., & Oliveira, M. B. P. P. (2008). Food authentication by PCR-based methods. European Food Research and Technology, 227, 649665. Mafra, I., Roxo, A., Ferreira, I. M. P. L. V. O., & Oliveira, M. B. P. P. (2007). A duplex polymerase chain reaction for the quantitative detection of cows milk in goats milk cheese. International Dairy Journal, 17, 11321138. Mafra, I., Silva, S. A., Moreira, E. J. M. O., Ferreira da Silva, C. S., & Oliveira, M. B. P. P. (2008). Comparative study of DNA extraction methods for soybean derived food products. Food Control, 19, 11831190. Maratagas, M., Skandamis, P., Nychas, G.-J. E., & Drosinos, E. H. (2007). Modelling and predicting spoilage of cooked, cured meat products by multivariate analysis. Meat Science, 77, 348356. Matsunaga, T., Chikuni, K., Tanabe, R., Muoya, S., Shibata, K., Yamada, J., et al. (1999). A quick and simple method for the identication of meat species and meat products by PCR assay. Meat Science, 51, 143148. Papadima, S. N., Arvanitoyannis, I., Bloukas, J. G., & Fournitzis, G. C. (1999). Chemometric model for describing Greek traditional sausages. Meat Science, 51, 271277. Pappa, I. C., Bloukas, J. G., & Arvanitoyannis, I. S. (2000). Optimization of salt, olive oil and pectin level for low-fat frankfurters by replacing pork backfat with olive oil. Meat Science, 56, 8188. Rodrguez, M. A., Garca, T., Gonzlez, I., Hernndez, P. E., & Martn, R. (2005). TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. Meat Science, 70, 113120. Sawyer, J., Wood, C., Shanahan, D., Gout, S., & McDowell, D. (2003). Real-time PCR quantitative meat species testing. Food Control, 14, 579583. Somma, M. (2006). Extraction and purication of DNA (session 4). In M. Querci, M. Jermini, G. V. Eede (Eds.), Training course on the analysis of food samples for the presence of genetically modied organisms. European Commission DG-JRC. <http://gmotraining.jrc.it>. Sultan, K. R., Tersteeg, M. H. G., Koolmees, P. A., de Baaij, J., Bergwerff, A. A., & Haagsman, H. P. (2004). Western blot detection of brain material in heated meat products using myelin basic protein and neuron-specic enolase as biomarkers. Analytica Chimica Acta, 520, 183192.

Fig. 6. Regression analysis between the predicted pork meat percentage and the actual value using the software SPSS for Windows (version 17.0). Y = 0.98X + 1.45, R2 = 0.99, n = 34.

cate assays, veried by the low coefcients of variation (from 4.08% to 7.61%), demonstrates the high reproducibility of the developed methodology. With these data, a linear regression with 95.0% individual prediction interval was performed between actual and predicted values. Data in Fig. 6 shows that all the values were included in the established interval. The correlation between the estimated and actual values was 0.99 with a slope very close to 1 (0.98), which conrms the high proximity between the predicted and actual values (Fig. 6). This result demonstrates the applicability of the established technique for estimating the addition of pork in poultry meat products. 4. Conclusions Nowadays, species identication in foods of animal origin is a challenge for the food control laboratories, being of utmost importance to guaranty the enforcement of EU legislation. Thus, there is a need for reliable, fast and sensitive methodologies that ensure an effective detection of frauds in the meat sector by the identication of species present in the meat products. In the present work, a duplex methodology proved to be a reliable and sensitive tool for the detection of pork in poultry meat with a sensitivity of 0.1% using a 35-cycle duplex PCR. The quantication of pork meat addition was also proposed using a 25-cycle duplex PCR in the range 175% by means of a normalised calibration curve. The applicability of the technique was demonstrated by the in-house validation using blind binary meat mixtures. The obtained results showed that the proposed methodology can be effectively used for estimating pork addition in poultry meat, representing a lower cost, reliable, specic and sensitive alternative to real-time PCR. References
Aida, A. A., Man, Y. B. C., Wong, C. M. V. L., Raha, A. R., & Son, R. (2005). Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. Meat Science, 69, 4752.