Meat Science 82 (2009) 444–449

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

Identification of meat species by TaqMan-based real-time PCR assay

Z. Kesmen a,*, A. Gulluce a, F. Sahin b, H. Yetim a
a
Erciyes University, College of Engineering, Food Engineering Department, 38039 Kayseri, Turkey
b
Yeditepe University, College of Engineering, Bioengineering Department, 34755 Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a convenient, sensitive and specific real-time PCR assay was described for the species iden-
Received 3 December 2008 tification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes
Received in revised form 19 February 2009 were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respec-
Accepted 20 February 2009
tively, and the performance of the method was tested. In the results, no cross-reaction was observed
between the donkey and pork species specific primer-probe systems and non-target species (bovine,
ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific pri-
Keyword:
mer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-
Meat species identification
Real-time PCR
time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA
TaqMan probe from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be
Horse suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method
Donkey for the routine meat species identifications studies in raw or cooked meat products.
Pork Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
In order to avoid unfair market competition and protection of
consumers from false labeled meat products that usually accepted
for economical, religious and health reasons require development
of a reliable and sensitive analytical tools that facilitate routine
control tests of meat species in different foods and feedstuffs. For
this purpose, numerous analytical methods have been developed
for the species identification of animal tissues in meat products.
These methods that based on the detection of species-specific pro-
teins, and related techniques like electrophoresis (Cota-Rivas &
Vallejo-Cordoba, 1997; Zerifi, Labie, & Benard, 1992), isoelectric
focusing (IEF) (Kim & Shelef, 1986; King, 1984; Skarpeid, Kvaal, &
Hildrum, 1998), and enzyme-linked immunosorbent assays (ELISA)
(Andrews, Berger, Mageau, Schwab, & Jhonston,1992; Berger,
Margeau, Schwab, & Johnston, 1988; Chen & Hsieh, 2000). These
methods have proven to be inadequate or less sensitive for the spe-
cies identification of meat products that have been exposed to very
high cooking temperatures causing denaturation of the proteins
(Guoli, Mingguang, Zhijiang, Hongsheng, & Qiang, 1999; Jemmi &
Schlosser, 1991; Koh, Lim, Chua, Chew, & Phang, 1998; Meyer, Can-
drian, & Lüthy, 1994). However, methods of DNA analysis based on
polymerase chain reaction (PCR) offer a potential for the precise
detection of the animal species used, even for the products that
have been subjected to intensive heat processing and products
with complex composition (Chikuni, Tabata, Kosigiyama, &
* Corresponding author. Tel.: +90 352 437 4937/32729; fax: +90 352 437 5784.
E-mail address: zkesmen@erciyes.edu.tr (Z. Kesmen).
Monma, 1994; Kesmen, Sahin, & Yetim, 2007; Meyer et al.,
1994). Conventional PCR techniques allow qualitative detection
of the different animal species in a mixture, but they are not appro-
priate to make quantification of the animal tissue in the product.
The most recent reports have focused on the use of real-time PCR
for meat species identification and quantification (Lahiff et al.,
2002; Laube et al., 2003; Mendoza-Romero et al., 2004; Sawyer,
Wood, Shanahan, Gout, & McDowell, 2003). Real-time PCR is
widely accepted as a robust assay for the species identification
and quantification of nucleic acid molecules due to its higher sen-
sitivity and specificity, larger dynamic range of detection, and less
carry-over contamination risk. In the quantitative real-time PCR
(qPCR) technique, amplification of the target gene is monitored
by an increased fluorescence signal which enables direct assess-
ment of the results after the PCR application without additional
detection steps. Currently, two formats correlating the PCR prod-
ucts with the fluorescent signal are available. The first method is
to use intercalating fluorescent dyes like SYBR Green (López-And-
reo, Garrido-Pertierra, & Puyet, 2006; Walker et al., 2003). This
type of dyes bind to all double-stranded DNA present, including
any non-specific PCR products and the primer-dimer complex
due to the specificity only depending on two PCR primers. Sec-
ondly, the more accurate and reliable method is to use fluorescent
reporter probes. This method utilises an additional primer, the
probe, which also binds specifically to the target DNA sequence
during the annealing step. The probe-based chemistries is com-
monly based on the use of a TaqMan fluorogenic probe system
(Brodmann & Moor, 2003; Chisholm, Conyers, Booth, Lawley, &
Hird, 2005; Dooley, Paine, Garrett, & Brown, 2004; Hird, Chisholm,

0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2009.02.019
 Z. Kesmen et al. / Meat Science 82 (2009) 444–449
& Brown, 2005; Hird et al., 2004; Krcmar & Rencova, 2005; Rodri-
guez, García, González, Hernández, & Martín, 2005; Zhang, Fowler,
Scott, Lawson, & Slater, 2007). TaqMan probes have an fluorescent
reporter dye at one end and a quencher dye that inhibits fluores-
cence at the other end. During the extension stage, the probe is
broken apart by the DNA-polymerase and begins to fluorescence
with more intensity.
Real-time PCR based assays have previously been reported by
some researcher for the identification of animal derived material
in meat mixtures by amplifying mtDNA genes as 12 S rRNA (Rodri-
guez et al., 2005), cytochrome b gene (Chisholm et al., 2005; Doo-
ley et al., 2004; Hird et al., 2004), and 16S rRNA (Sawyer et al.,
2003). However, no report has been published for the real-time
PCR technique targeting the mitochondrial ND2 and ND5 genes
for the purpose of species identification. The aim of this work
was to evaluate the performance of the specific primers for each
species. TaqMan probes are designed to determine and quantify
horse, donkey, and pork species in both raw and cooked meat
complexes.
2. Materials and methods
2.1. Preparation of the meat patty samples
In the preparation of meat patty samples, binary meat mixtures,
containing each species of pork, horse or donkey meat ranged from
0.0001% to 10% levels (corresponding range of 0.0001–10 ng) with-
in a beef mixture were used. In order to dilute the target meats in
the patties, beef was added to the mixture at approximately 50%
level of the batch and blended for 2 min to obtain a thoroughly
homogeneous meat mixture each time. In this way, the errors that
usually occur during blending were eliminated.
Various ingredients, red pepper (0.7%) black pepper (0.7%),
onion (7%) and salt (2%) were added to the meat mixtures, and
an average of 20 g of meat patties were prepared from each mix-
ture. After preparation of the raw patties, the samples were cooked
in a commercial electric grill set at 180 ± 5 °C which resulted in an
internal temperature of 80–85 °C after cooking for 5 min. Then the
DNA isolation was made from the raw and cooked meat patty
samples.
2.2. DNA isolation
In order to have representative specimen, approximately 10–
15 g raw and cooked meat patty samples and pure meats from each
species investigated were sampled and then powdered with liquid
nitrogen after holding at 80 °C overnight. The total DNA was ex-
tracted from 25 mg of powdered samples using a commercial kit
(Qiagen, Hilden, Germany) according to manufacturer’s protocol.
DNA concentration was measured by spectrophotometric analysis
(Heptinstall & Rapley, 2002).
2.3. Primer design
Species specific primers and TaqMan probes for the detection
of pork, horse and donkey meat DNAs were designed from different
regions of the mitochondrial genome, following the alignment of
available sequences from GenBank database with (version 1.8) of
Clustal W. (Higgins, Bleasby, & Fuchs, 1992). Using Primer Express
software, primer and probe sets were designed to have mismatches
to all other species at the 30 position of either, or where possible,
both sense and antisense primers. Most primer design programs
take only one target sequence without considering mispriming to
off-target templates and therefore, one must manually compare
the aligned sequences and then start from the 30 end of the primer
445
on a point of mismatch prior to running Primer Express software to
find the specific primer probe sets. Using this approach, we de-
signed primer and probe sets to be specific for horse, donkey and
pork when compared with other commonly raised livestock spe-
cies (Fig. 1). The species specific region of ATPase6/ATPase8 gene
in horse, ND2 (NADH dehydrogenase subunit 2) gene in the donkey
and ND5 (NADH dehydrogenase subunit 5) gene in pig DNA were
amplified and the primers generated species specific fragments
of 147, 183 and 115 bp length for horse, donkey and in pork,
respectively. Additionally, common sense and antisense primers
and probe for mammalian were designed to amplify a conserved
region of 74 bp of 16 sRNA gene as a control of amplificability
and false negativity on PCR results. Primers and probes, labeled
50 -carboxyfluorescein (FAM) and 30 fluorescent quencher (TAMRA)
were purchased from TibMolbiol.
2.4. PCR primers specificity and sensitivity test
The specificity of each species specific primer was confirmed by
amplification of 100 ng of horse, donkey, pork, bovine, ovine, chick-
en and turkey genomic DNA, and a negative control was without
DNA. Each assay was tested against DNA from all seven species
i.e. against its target species and the six remaining species to con-
firm assay specificity. In the determination of the detection limit of
the specific primers and probes, a 1:10 serial dilutions of horse,
donkey and pork genomic DNAs were prepared. For each target
species, 100, 10, 1, 0.1, 0.01, 001 and 0.0001 ng DNA was separately
added to the per 25 ll reaction mixtures to construct a standard
curve.
2.5. Real time PCR assay
The TaqMan PCR reaction was performed in a final volume of
25 ll using 12.5 ll of Quantitect Probe PCR mix (Qiagen, Hilden,
Germany) 100 ng DNA, 0.8 lM of each primer and 0.25 lM of the
TaqMan probe. The amplifications were performed on a Line Gene
II PCR device (Bioer Technology Co., Hangzhou, China) and thermal
cycling protocol of 95 °C for 15 min followed by 40 cycles of 95 °C
for 15 s and a temperature 60 °C for 1 min was applied. Reactions
were replicated at least twice per experiment and experiments
were replicated three times to verify results. Then the product
amplifications were confirmed using standard agarose gel tech-
niques (Sambrook, Fritsch, & Maniatis, 1989).
3. Results
3.1. Specificity of the TaqMan probe systems
In this research, for the determination of horse, donkey and por-
cine tissue in the meat mixtures, TaqMan probes were designed
specific to each species, and the amplification conditions were
optimized by using these primers. The specificity of the primers
and probes were tested for 7 livestock species; pork, horse, donkey,
bovine, sheep, chicken and turkey. It was determined that while
the primers and probes specific to the species of donkey and pig
showed no cross-reaction with any of the non-target species, that
of the horse showed cross-reaction with the pig species after the
33 cycle (corresponding to 0.01 ng horse DNA). The ability of the
amplification of the DNA’s from 7 species examined to eliminate
the false negative results was tested with a primer and probe set
that was common to both mammalian and poultry. With this com-
mon probe and the primer set for genomic DNA (100 ng) that be-
long to the species of horse, donkey, pig, bovine, ovine, chicken
and turkey, the ct values of 15.60, 15.14, 14.68, 17.86, 15.10 and
18.36 were calculated, respectively.
446 Z. Kesmen et al. / Meat Science 82 (2009) 444–449

Fig. 1. Sequence alignment of the PCR products for each species investigated and most commonly consumed meat species. (Accession number of bovine, ovine, chicken,
turkey, horse, donkey and pork are AY526085, AF010406, X52392, EF153719, X79547, X97337 and AF034253, respectively).

3.3. Species determination and quantification with TaqMan assay in

Table 1
meat patties
Ct values for each species tested with the species specific primer-probe sets.

Species DNA concentration Ct values of species specific primer-probe In the samples of raw and cooked meat patties made with bin-
(ng) sets
ary meat mixtures, the detection limit was 0.0001% (corresponding
Horse Donkey Pork 0.0001 ng target DNA). No difference has been observed between
Target species 0.0001 36.84 37.28 37.37 the Ct values of raw and cooked samples. Moreover, the ingredi-
0.001 34.87 33.21 33.8 ents used in the preparation of the samples had no inhibitor effect
0.01 31.34 30.68 31 on real-time PCR. Also, no adverse effect of heat treatment, pro-
0.1 27.88 27.47 28
1 23.96 24.05 24.78
cessing conditions and ingredients used for meat patty preparation
10 21.04 20.88 21.15 was noticed in the PCR amplification. It has been determined that
Horse 100 17.25 40.00 40.00
the Ct values corresponding to the DNA concentration of target
Donkey 100 40.00 17.53 40.00 species showed a linear change in all the patty samples, and for
Pork 100 33.01 40.00 17.69 all the samples, the correlation coefficient changes was between
Bovine 100 40.00 40.00 40.00 0.981and 0.995.
Ovine 100 40.00 40.00 40.00
Chicken 100 40.00 40.00 40.00
Turkey 100 40.00 40.00 40.00 4. Discussion

Real time PCR technique is a very robust technique on species

3.2. Sensitivity and linearity of TaqMan probe system
To determine the sensitivity and linearity of the real-time PCR
technique, the genomic DNA obtained from each target species
starting from the 100 ng (100%) target DNA, 10 fold serial
dilutions were prepared and the Ct responses were determined.
With all three species, horse, donkey and pork, a sensitivity of
at least 0.0001 ng DNA which corresponds to 0.0001% were
obtained. For each species, the sensitivity was determined as
0.0001 ng (Table 1). To test the system and asses the range of
real-time PCR linearity, a standard curve were constructed on
genomic DNA from each target species. It has been observed that
the standard curve that was obtained by plotting the Ct values
that correspond to logarithmic DNA concentrations shows a linear
correlation (coefficient of correlation >0.998) in the range from
100 ng to 0.0001 ng for the each species that was tested (Fig. 2).
Additionally, the slopes for each species are close to the theorical
value (3.32), which can be achived with a PCR efficiency of
100%.
identification in both specificity and sensitivity. The specificity of
the method is related to the specificity of the first degree designed
primer and probe. Previous studies of species identification where
PCR technique was used on meat and meat products, target region
are chosen usually on mitochondrial DNA, because it shows an
adequate degree of intra and interspecies variability and also due
to the high number of copies found per cell (Kocher, Thomas,
Meyer, Edwards, & Pääbo, 1989). In general, cytochrome b (Dooley
et al., 2004; Matsunaga et al., 1999; Meyer, Höfelein, Lüthy, & Can-
drian, 1995), D-loop (Fei et al., 1996; Monteil-Sosa et al., 2000), 12S
rRNA (Rodriguez et al., 2004, 2005) and 16S rRNA (Borgo, Souty-
Grosset, Bouchon, & Gomot, 1996; Sawyer et al., 2003) genes have
been used for species identification. However, in this study, con-
trary to the previous target regions, species specific primers and
TaqMan probes were designed targeting the mitochondrial ND5
and ND2 for the identification of donkey and pork species. Because
the lengths of these genes are appropriate, their mutation degrees
are sufficient and they have sequence databases available for many
plant and animals (Herman, 2001). The regions showing a rela-
tively high degree of divergence among the species investigated
 Z. Kesmen et al. / Meat Science 82 (2009) 444–449 447

Fig. 2. Relationships between the amount of template DNA in the assay and Ct values of each species (amounts of DNA ranged from 0.0001 to 100 ng). (a) Ten fold dilution
series of horse DNA, (b) ten fold dilution series of donkey DNA, (c) ten fold dilution series of pork DNA.

were selected to design species specific probes and primers and
amplifying products shorter than 150 bp. Restriction on the PCR
amplicon size (maximum 150 bp) lend this system to the detection
of DNA in highly processed food products where likelihood of DNA
degradation is very high.
Additionally, another TaqMan PCR system was developed for
the amplification control to exclude probable false negative results.
Thus amplification control TaqMan probe system serves for the
purpose of checking the quality of nucleic acids extracted and
can be applied prior to the specific detection of horse, donkey
and pork. Using the common primer probe system, specific for
mammals and poultry, the DNA of all seven species investigated
could be amplified and clearly detected.
In this study, although a standard 40 cycles system was applied,
no cross reaction was observed between the primer and probe
systems specific to the donkey and pig species and the non-target
species tested. On the many real-time PCR applications, no cross-
amplification of non-target DNA is required. For this purpose,
two complementary strategies were used to confer specificity on
a real time PCR assay; first by placing the 30 end of the primers
on a point of sequence heterogeneity and second, by truncating
the primers at the 50 end to lower the calculated melting temper-
ature. Chisholm et al. (2005) have developed a TaqMan probe
method that targeted the mitochondrial cytochrome b gene to
identify the horse and donkey species. Researchers eliminated
the cross amplification problem caused by the high degree se-
quence homology between these two species by truncating of
primers at the 50 end. However, we know that truncating the prim-
ers at the 50 end increases the specificity, but it causes a decrease in
efficiency. For this reason, sequence heterogeneity is necessary for
specific and sensitive detection. For a specific detection, primer
specificity is the most important necessity which occurs as well
on the primer’s 30 end, the terminal mismatches prevents the elon-
gation of the Taq polymerase and thus impede any non-specific
amplification (Huang, Arnheim, & Goodman, 1992).
The differentiation of horse from donkey meat is challenging
since they are closely related species having a high degree of
sequence homology. Nevertheless, differentiation is desirable be-
cause horse is consumed in some countries while donkey is not.
In addition to this, no cross-amplification between horse and don-
key was observed in this work. Because the horse primer and probe
set had five base differences in the sense primer and probe, and
four bases differences in the antisense primer when compared to
the donkey sequences. Similarly the donkey primer and probe set
had six base differences in the sense primer, five base differences
in probe and four base differences in antisense primer when
448 Z. Kesmen et al. / Meat Science 82 (2009) 444–449

compared to the horse sequence. In this study, Ct 33.01 values Table 2

were obtained for the 100 ng pork DNA with the primer and probe Results of the experimental raw and cooked meat patties samples.

set specific only to the horse species. With the same primer and Assay target The ratio of target species Ct values from Ct values from
probe system, horse genomic DNA’s for 100 ng Ct 17.25 for level species in the binary mixture (%) the raw samples the cooked samples
of % 0.01 (corresponding for 0.01 ng) Ct 32 values were obtained. Horse 10 20.89 21.28
However, for species identification, it would be ambiguous as to 5 21.63 22.35
whether the signal was either due to presence of the analyte at a 1 23.77 24.71
0.5 23.86 24.76
low or there was the cross-amplifying species at a relatively high 0.1 25.86 26.51
level. Therefore, some researchers reported no cross reactivity be- 0.05 28.17 28.22
tween the specific primers and probes for each target species and 0.01 30.51 30.34
non-target species when a Ct value of 30 as the cut-off point was 0.005 31.86 31.44
0.001 34.14 33.46
used (Dooley et al., 2004).
0.0005 36.56 34.15
The aim of TaqMan probe system is to detect and quantificate 0.0001 38.09 37.95
the degraded DNA that belong to the target species on very low
Donkey 10 22.68 21.23
levels. In this work, all of the pure meat, raw or cooked samples 5 23.24 23.06
were determined with TaqMan probe system on 0.0001 ng 1 25.72 25.1
(100 fg) DNA level. This result is lower than the results of Rodri- 0.5 26.74 26.06
guez et al. (2005) who determined the detection limit as 0.01 ng 0.1 28.06 27.78
0.05 30.53 30.29
DNA meaning 0.1% using TaqMan probe system specific to pig spe-
0.01 32.31 33.1
cies on mitochondrial 12S rRNA, and this is also lower then the re- 0.005 35.00 33.67
sults of Chisholm et al. (2005) who developed a real time PCR assay 0.001 35.93 35.53
which detects the horse and donkey species in commercial prod- 0.0005 36.35 35.64
0.0001 36.81 36.68
ucts on the levels of 1 pg and 25 pg, respectively. Mendoza-Romero
et al. (2004) was able to determine the bovine DNA in very low le- Pork 10 21.54 21.95
5 22.5 22.94
vel, 10 fg with a real-time PCR technique which targets the rumi-
1 24.93 24.97
nant short interspersed nuclear element’s 88 bp fragments. 0.5 25.64 25.84
Meat species adulteration in ground and heat treated commi- 0.1 26.88 27.67
nuted meat products has been a widespread problem in food retail 0.05 29.67 29.90
market in the many areas of the world. A common adulteration of 0.01 32.19 31.78
0.005 32.69 32.57
meat products is addition of pork, donkey and horse meats which
0.001 34.94 34.84
are undesirable and/or cheaper meats in more expensive meats 0.0005 35.88 35.06
and undeclared on the products. In this work, these species have 0.0001 37.06 36.97
been selected and substituted in the meat patties that is widely
consumed in many countries, and also hamburger type of meat
products carries potential risk of adulteration. A cooking procedure for the preparation of this binary meat mixtures which were used
for a desirable organoleptic quality and for an acceptable level of in the production of patty samples. Therefore, the identification of
safety, the grill was set to 180 ± 5 °C in order to obtain an internal the target species was possible even in the lowest contamination le-
temperature of 85 °C internal temperature which requires approx- vel (0.0001%), and the correlation between the Ct values corre-
imately 5 min of heating time. The similarity of the Ct values ob- sponding to the logarithmic DNA concentration was very high
tained for the cooked and raw samples on the same (average 0.990) in both raw and cooked samples.
contamination level (Table 2) shows that the heating process does
not cause a high degree of DNA fragmentation like a heat treat- 5. Conclusions
ment under high pressure would. In addition to this, especially
the lengths of the target amplicons were so small (<150 bp) that The TaqMan probe real-time PCR assays evaluated in this study
no amplification problems caused by intense DNA fragmentations have a high potential as a molecular tools that can be used in rapid
could occur. Laube et al. (2003) reported that the ingredients used and routine detection and quantization of horse, donkey and pig
in the processed meat products could contain PCR inhibitors iso- present in the raw and heat treated ground meat mixtures with a
lated together with the nucleic acids and these can cause false-neg- single reaction step of PCR. By means of the optimized assay, the tis-
ative results by inhibiting DNA polymerase enzymes. However, in sue of each species could be distinguished from the phylogenetically
this work, it was determined that the ingredients used in the pro- close species and/or other livestock animals, and the presence of a
duction of the meat patty samples did not have an inhibitor effect. 0.1 pg template DNA in a water solution might be detected. Conse-
In this research, a commercial isolation kit was preferred to min- quently, the TaqMan probe real-time PCR based assay can be recom-
imize the errors that can be caused by the DNA isolation procedure. mended for the detection of animal tissue by food control agency or
Because it has been noted that the extraction method based on the laboratories, and it might be a reliable and practical method for the
binding of DNA to a silica matrix in the presence of chaotropic determination of technically inevitable contamination and/or
agents is more efficient, and it removes the PCR reaction inhibitors intentional admixtures in highly processed meat products.
(Di Pinto, Forte, Conversano, & Tantillo, 2005). Again, it has been
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