Documenti di Didattica
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Documenti di Cultura
Food Control
journal homepage: www.elsevier.com/locate/foodcont
Laboratrio de Gentica Animal, Departamento de Zootecnia, Escola de Veterinria, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Valid Biotechnology research team, INOVA, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
c
Escola de Engenharia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
d
Departamento de Tecnologia e Inspeo de Produtos de Origem Animal, Escola de Veterinria, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 March 2012
Received in revised form
15 May 2012
Accepted 22 May 2012
Fraudulent species substitution in food products is a reality in many markets around the world. The
consequences are serious and impact governments, consumers and producers. Molecular methods,
including PCR-based techniques, are available to prevent species substitutions and to authenticate food
content. Here, we describe a method for calculating the bovine and buffalo content in milk- and meatderived food products. The method applies real-time PCR using primers designed to specically amplify
bovine or buffalo DNA. The amplication efciencies of both primer sets were assessed using the TaqMan
and SYBR Green systems. Both sets of primers showed satisfactory results. A quantication procedure is
proposed and involves amplifying a sample with both primer sets and then normalizing the total DNA using
the total non-normalized bovine and buffalo DNA. To correct for the potential deviations between the real
and measured DNA quantity caused by biological differences between species, we propose the use of calibration curves generated from each analyzed matrix. These curves include a set of controlled admixtures of
bovine and buffalo material. Using this method for dairy samples containing known bovine and buffalo
content, the use of the calibration curve always approximated the measured to the expected quantities. This
technique was then tested on commercial samples. Our method was efcacious and reliable and can be
applied to the routine analysis of these products. Although we only tested the methodology on dairy
products, it can also be applied to other food matrices, such as meat-derived products. A patent document for
the technique has been submitted to the Brazilian Patent Ofce (INPI e Instituto Nacional da Propriedade
Industrial e www.inpi.gov.br) under the number 014110002844.
2012 Elsevier Ltd. Open access under the Elsevier OA license.
Keywords:
Food authentication
Bovine
Buffalo
Real-time PCR
Quantication
1. Introduction
The adulteration of foods with material from other species with
greater availability and/or lower cost is observed worldwide. An
example of this process is the addition of bovine milk to sheep, goat
and water buffalo dairy products (Herman, 2001). Other animal
products, such as seafood and meat-derived products, are also
subjected to content substitution (Carvalho, Pimenta-Neto, Brasil, &
Oliveira, 2011; Gonzlez-Crdova, Caldern de la Barca, Cota,
& Vallejo-Crdoba, 1998; Lahiff et al., 2001; Soares, Amaral,
132
2. Methodology
Pure dairy and meat samples derived from bovine (Bos taurus
taurus or Bos taurus indicus) and buffalo (Bubalus bubalis) material
were used to test the efciency of the primer/probe sets (described
in section 2.4). These samples were also applied to create a calibration curve (section 2.6) and to test the technique presented here
(section 2.7). Commercial samples were acquired from local
markets to test the usefulness of the method.
Table 1
Primer/probe sets used in this study.
Primer/probe
Typea
Sequence (50 / 30 )
Target
BosF
BosR
MamCytB
Bub2F
Bub2R
Bub16S
Forward primer
Reverse primer
TaqMan e MGB probe
Forward primer
Reverse primer
TaqMan e MGB probe
CGGAGTAATCCTTCTGCTCACAGT
GGATTGCTGATAAGAGGTTGGTG
FAM e CATGAGGACAAATATC e MGBb
TCAGCCCAAAGAAAAATAAACCA
GTCACCCCAACCGAAACTGT
FAM e TAAGGARTAACAACAMTCT e MGBc
CytB (bovine)
CytB (bovine)
CytB (mammals)
16S (buffalo)
16S (buffalo)
16S (mammals)
a
b
c
Only the primer sets were used with the SYBR Green system.
In the test, the FAM uorophore was used to target the probes. Other uorophores can be used in its place.
The Bub16S probe was designed to undergo degeneration. According to IUPAC, R A or G, M A or C.
CtBos
=2
CtBos
CtBub
(1)
or
NBos 2CtBos
(3)
and
NBub 2CtBub :
133
(4)
134
Table 2
Data from efciency experiments using Bos and Bub amplication sets.a
PCR system
Primer/probe
set
Sample
material
Efciency (%)
Slope
R2
Ct range
Initial DNA
amount range
Number of dilution
points in the curve
Specicity
limit (Ct)
TaqMan
Bos
Bos
Bub
Bub
Bos
Bub
Meat
Cheese
Meat
Cheese
Cheese
Cheese
96.0
91.6
90.1
92.7
93.1
92.9
3.42
3.54
3.58
3.51
3.49
3.50
0.99
0.99
0.99
0.97
0.99
0.98
25.2e30.5
16.8e24.0
28.7e32.9
17.8e25.2
16.7e22.0
15.7e20.0
0.098e0.003 ng
10e0.08 mg
0.08e0.005 ng
10e0.08 mg
1.25e0.04 mg
1.25e0.08 mg
6
8
5
8
6
5
37
31
35
35
40
40
SYBR Greenb
a
b
Each variable was measured as described in materials and methods section 2.3.
The SYBR Green system was used to verify the amplication efciency of both primer sets on dairy samples to illustrate the versatility of the technique.
2010 (Fig. 2). The measured R2 value (0.998) showed that the
points were well represented by the straight trend line. The derived
trend line equation was then applied to the following experiments
using an automated spreadsheet (Calibration Software). This step
allowed us to correct the measured to the real quantity of the
bovine and buffalo material present in a sample.
3.4. Method validation
Admixtures of pure bovine and buffalo dairy samples were again
tested in controlled ratios. DNA was extracted and amplied from
these samples using the methodology described above for TaqMan
system. The Calibration Software was then used to calculate the real
bovine content in each sample. As shown in Table 3, the quantication method was effective in determining the real bovine content
of a given sample. The accuracy of the method was very high. The
measured value was at most 12.5% greater or less than the expected
value (e.g., expected 0.16 / measured 0.14). Importantly, the
calibration curve successfully corrected the calculated quantication to the expected value in all cases (Table 3).
Commercial samples purchased from local retailers were also
analyzed using our method (Table 4) to show its applicability to the
routine analysis of commercial buffalo products. Controls with 50,
100 and 0% Bos/Bos Bub content were tested with each experimental sample. A maximum of 6% deviation from the expected
value was observed for these controls (e.g., expected
0.5 / measured 0.47).
Fig. 1. Expected and measured bovine content in the diverse ratios of controlled mixtures of two calibration curves. Known amounts of bovine and buffalo cheese pure samples
were weighed and mixed to create ratios of controlled contamination for two calibration curves. DNA from the resulting admixtures was extracted and amplied using the Bos and
Bub primer/probe sets and the TaqMan system. Equation (1) was used to calculate the proportion of the bovine content in each sample.
135
Table 4
Proportion of Bos content in commercial samples.
Fig. 2. Calibration curve used to correct the error in the measured bovine quantity. The
mean bovine quantity measured for the different ratios in the two controlled
contamination experiments was plotted against the expected Bos content. A trend line
was added using Microsoft Excel 2010. The trend line equation was applied in the
following experiments to adjust for the real bovine content of buffalo-derived dairy
samples. R2 was calculated to assess the t of the trend line to the measured points.
4. Discussion
Several methods are currently being applied to authenticate
food products, as reviewed by Reid, ODonnell, and Downey (2006).
However, the complexity of food matrices and small quantities of
the contaminant present challenges to the sensitivity of the available techniques (Vallejo-Cordoba & Gonzlez-Crdova, 2010).
Among the DNA-based technologies, PCR was chosen for the
present study because it can quickly and specically amplify small
DNA quantities (Mafra et al., 2007) e even after heavy food processing (Rodrguez-Ramrez, Gonzlez-Crdova, & Vallejo-Cordoba,
2011). Mitochondrial DNA was chosen as the amplication target
because of its relative cellular abundance. This attribute increases
assay sensitivity (Lopparelli et al., 2007). Furthermore, the genetic
regions that were targeted are relatively small (approximately
100 bp). Thus, even DNA molecules degraded by the thermal,
chemical and/or physical processes through which food is prepared
can be detected.
Real-time PCR is commonly used to measure the quantity of
a DNA template in a given sample. Several methods are used to
calculate DNA quantity based on real-time PCR Ct results. One such
method is the relative quantication using comparative Ct (Livak &
Schmittgen, 2001). For this method, an internal control must be
applied to normalize the DNA quantities in different samples (Livak
& Schmittgen, 2001). In the majority of the studies that use realtime PCR to quantify contaminant species, normalization is
Table 3
Proportion of expected and measured Bos content in controlled contamination
experiments.
Ratios
Expecteda
Measuredb
After correctionc
0%
7%
16%
30%
50%
66%
78%
87%
97%
100%
0.00
0.07
0.16
0.30
0.50
0.66
0.78
0.87
0.97
1.00
0.00
0.07
0.13
0.30
0.46
0.65
0.75
0.85
0.96
1.00
0.00
0.07
0.14
0.30
0.47
0.66
0.76
0.86
0.97
1.00
Expected Bos content based on the bovine material added to the mixture.
Proportion of Bos content calculated using the method described in this
manuscript and Equation (1).
c
Corrected proportion of Bos content adjusted using the calibration curve.
b
Sample
Measureda
After correctionb
1
2
3
4
5
6
7
8
9
10
0.00
0.00
0.05
0.25
0.39
0.04
1.00
0.00
0.00
0.00
0.00
0.01
0.06
0.25
0.40
0.05
1.00
0.00
0.00
0.00
a
Proportion of Bos content as calculated using the method described in this
manuscript and Equation (1).
b
Proportion of Bos content corrected using the calibration curve.
136
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