Food Control 25 (2012) 666e672

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Food Control
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A comparative study of DNA extraction methods to be used in real-time PCR based

quantication of ochratoxin A-producing molds in food products
Alicia Rodrguez a, Mar Rodrguez a, M. Isabel Luque a, Annemarie F. Justesen b, Juan J. Crdoba a, *
a
b

Food Hygiene and Safety, Faculty of Veterinary Science, University of Extremadura, Avda. de la Universidad, s/n. 10071-Cceres, Spain
Department of Integrated Pest Management, Faculty of Agricultural Sciences, University of Aarhus, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 June 2011
Received in revised form
2 December 2011
Accepted 10 December 2011

Quantication of ochratoxin A (OTA)-producing molds in foods by real-time quantitative PCR (qPCR) may
be affected by the DNA extraction method used. In the present work, 6 different methods for extraction
of DNA from ochratoxigenic molds in foods were tested. Several combinations of mechanical and thermal
lysis of conidia with commercialized DNA extraction kits and enzymatic treatments or resins were
evaluated. DNA recovery and quality of extracted DNA was measured by testing the extracted DNA with
a conventional PCR and an SYBR Green qPCR amplifying the b-tubulin gene and the non-ribosomal
peptide synthetase gene, otanpsPN. Inhibition of conventional and qPCR was not observed when the
DNA-extraction method includes an initial thermal disruption of conidia before use of commercialized
extraction kit or resin, enzymatic treatment and/or lysis buffer. Of the six methods tested, the one
combining thermal lysis of conidia followed by a short enzymatic treatment and incubation with Chelex100 resin and nal extraction with the EZNA kit was selected, since the extracted DNA showed good
amplication by conventional PCR for b-tubulin gene and the highest DNA recoveries when tested by
qPCR. The method was subsequently validated in different food products such as ripened foods, nuts, and
grapes inoculated with Penicillium and Aspergillus species. With this Chelex100-enzymatic-EZNA method
good DNA recoveries ranging from 69 to 99% were obtained for all food matrices and fungal species
tested. This fast method is a promising tool to be used as routine analysis in HACCP systems in the food
industry for quantifying OTA-producing molds by qPCR.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Ochratoxin A
Mold
DNA recovery
DNA extraction
qPCR

1. Introduction
Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, carcinogenic, inmunotoxic, genotoxic and teratogenic effects (Abouzied
et al., 2002; Selma, Martnez-Culebras, & Aznar, 2008). Several
strains of Penicillium and Aspergillus spp. have been reported as OTA
producers (Dachoupakan et al., 2009; Lund & Frisvad, 2003; LpezMendoza, Crespo-Sempere, & Martnez-Culebras, 2009; Mul,
Susca, Logrieco, Stea, & Visconti, 2006; Sartori et al., 2006). OTAproducing strains may contaminate ripened foods (Bogs, Battilani,
& Geisen, 2006; Kure, Skaar, & Brendehaug, 2004; Nez,
Rodrguez, Bermdez, Crdoba, & Asensio, 1996), nuts (Magnoli
et al., 2007), wine and grape products (Atoui, Mathieu, & Lebrihi,
2007; Selma et al., 2008), among others. Furthermore, OTA has
been detected in these food products as a consequence of the

* Corresponding author. Tel.: 34 927 257 125; fax: 34 927 257 110.
E-mail address: jcordoba@unex.es (J.J. Crdoba).
URL: http://higiene.unex.es/
0956-7135/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.12.010

growth of these toxigenic mold species (Atoui et al., 2007; DallAsta

et al., 2008; DallAsta et al., 2010; Iacumin et al., 2009).
Early detection of OTA-producing species in food production,
even before fungal development, is crucial to prevent production of
OTA during the ripening process, nuts storage or grape harvest time
(Atoui et al., 2007; Magnoli et al., 2007), since there is no current
industrial process to detoxify foods contaminated with OTA. DNAbased methods such as conventional PCR and real-time quantitative PCR (qPCR) are a good alternative to traditional culturing
techniques and morphological identication, since they are rapid,
sensitive and specic (Gil-Serna, Vzquez, Sardias, Gonzlez-Jan,
& Patio, 2009). However, the validity and robustness of the results
obtained from these techniques depend on the efcient recovery of
fungal DNA from the food samples.
The quality of DNA extracted from foods is critical because the
efciency of conventional PCR and qPCR methods can be reduced
by the inhibitors from the matrix (Di Pinto et al., 2007; Lucero
Estrada, Velzquez, Di Genaro, & Stefanini de Guzmn, 2007;
Mafra et al., 2008). Several organic compounds such as polysaccharides or fatty acids from ripened foods (Makhzami et al.,

A. Rodrguez et al. / Food Control 25 (2012) 666e672

2008), nuts oils or polyphenols (Passone, Rosso, Ciancio, &

Etcheverry, 2010; Yu, Ahmedna, & Goktepe, 2004) and polysaccharides or tannins from grapes or wine grapes (Selma et al.,
2008) may act as inhibitors of PCR. PCR inhibition can be tested
with conventional PCR but the qPCR furthermore offers the
opportunity to estimate the DNA recovery (Jara, Mateo, Guillamn,
Torija, & Mas, 2008). A good DNA extraction protocol is essential to
avoid the presence of inhibitors from the food matrix or degradation and damage of DNA (Miller, Bryant, Madsen, & Ghiorse, 1999;
Ongol, Tanaka, Sone, & Asano, 2009).
Several DNA extraction methods have been developed to obtain
DNA from vegetable food matrices for the quantitative detection of
some specic toxigenic fungal species by qPCR (Fredlund et al.,
2008; Haugland, Brinkman, & Vesper, 2002). However, an efcient DNA-extraction method suitable to be used in quantication
of OTA-producing molds in different food matrices by qPCR has not
previously been reported.
The aim of this work was to evaluate by a comparative study, the
efciency of different DNA extraction methods developed by
combining traditional DNA extraction treatments, procedures and
kits. The most efcient DNA extraction method was selected and
validated in different food matrices to obtain DNA suitable for
sensitive quantication by qPCR of OTA-producing molds.
2. Material and methods
2.1. Fungal strains and OTA production
Two OTA-producing mold reference strains (Penicillium nordicum CBS 110769 and Aspergillus westerdijkiae CECT 2948) were used
in this study. Production of OTA of these two strains was conrmed
by micellar electrokinetic capillary electrophoresis (MECE) (Martn,
Jurado, Rodrguez, Nez, & Crdoba, 2004), and also by highpressure liquid chromatography-mass spectrometry (HPLC-MS)
(Sosa et al., 2002) after growing the mold strains in potato dextrose
agar (Sharlau Chemie S.A., Barcelona, Spain) for 15 days at 25  C.
2.2. Preparation of mold spores and inoculation of food matrices
Both mold strains were 3-point inoculated on Malt Extract Agar
and incubated for 20 days at 25  C. Spores were harvested by
ooding 3 plates with 5 mL of sterile nanopure water containing
10% glycerol (Scharlau Chemie S.A.), and rubbing the surface with
a glass rod. The conidial suspension was ltered through Whatman
paper No 1, diluted in sterile nanopure water, as necessary, and
quantied by microscope, using a Neubauer counting chamber.
Non-sterile commercial foods (Iberian dry-cured ham, ripened
cheese, peanut, pistachio and grape) were inoculated in sterile bags

667

to a nal concentration of 2.5  103 spores of the OTA-producing

molds per gram of food matrix. The natural fungal contamination
of the uninoculated food samples was tested and was always lower
than 1 log cfu/g. In addition, DNA of this fungal contamination was
tested by qPCR for OTA-producing molds as described below and
was always negative.
2.3. DNA extraction protocol
DNA extraction methods were evaluated using the Iberian drycured ham as food matrix and P. nordicum CBS 110769 as OTAproducing mold. The effect of different steps in the DNA extraction procedure such as type of dilution buffer, method for breakage
of conidia, type of commercial DNA extraction kit, DNA extraction
solvents and DNA precipitation alcohols were tested (Table 1). Thus,
six DNA extraction methods were assayed on inoculated Iberian
dry-cured ham and on the conidial suspension. An overview of
these methods is given in Table 1.
In all methods assayed, 5 g of inoculated Iberian dry-cured ham
were homogenized with 10 mL of TriseHCl buffer (pH 8.0) in a lter
bag BagPage (Interscience, Paris, France) using a pulsier equipment (Microgen bioproducts, Surrey, UK). The ltrate was transferred to a sterile tube and processed in two different ways: (a) it
was centrifuged at 13,000 rpm for 10 min and pellets were treated
using the corresponding protocols (methods 1e4) and, (b) directly
according to extraction methods 5 and 6. All methods were assayed
in duplicate.
2.3.1. Mechanical breakage of conidia and EZNA kit (MechanicalEZNA method)
In this method, 20 mL of liquid nitrogen was added to the pellet,
which was ground to a ne powder in a 80  C prefrozen mortar
and pestle for 2 min. The crude extract was processed according to
instructions of the commercial DNA extraction kit EZNA Fungal
DNA Mini Kit (Omega bio-teck, Doraville, GA, USA).
2.3.2. Thermal breakage of conidia and EZNA kit (EZNA method)
Pellets were resuspended in 100 mL sterile nanopure water,
boiled (95  C for 10 min) to release the DNA, and cooled on ice for
10 min. The crude extract was processed according to instructions
of the commercial DNA extraction kit EZNA Fungal DNA Mini Kit
(Omega bio-teck). DNA precipitation was carried out in two
different ways: with pure isopropanol (option 2a) and with isopropanol:ethanol (2:1) (option 2b).
2.3.3. Thermal breakage of conidia and QIAGEN kit (QIAGEN method)
In this method, pellets were treated in the same way as Section
2.3.2. Then, the crude extract was processed according to

Table 1
General description of DNA extraction methods evaluated in this study.
Method name
1. Mechanical-EZNA
2. EZNA
3. QIAGEN
4. CTAB-EZNA

Description
2a (isopropanol precipitation)
2b (isopropanol:ethanol precipitation)
4a (without 2-mercaptoethanol)
4b (with 2-mercaptoethanol)

5. Chelex100-EZNA

5a (EZNA kit)
5b (CTAB and EZNA kit)
5c (CTAB, microwave and EZNA kit)

6. Chelex100eenzymatic-EZNA

Mechanical cell lysis EZNA kit

Thermal cell lysis EZNA kit DNA precipitation by isopropanol
Thermal cell lysis EZNA kit DNA precipitation by isopropanol:ethanol
Thermal cell lysis QIAGEN kit
Thermal cell lysis CTAB enzymatic digestion (proteinase K and RNase)
chloroform EZNA Kit.
Thermal cell lysis CTAB enzymatic digestion (proteinase K and RNase)
2-mercaptoethanol chloroform EZNA Kit.
Chelex-100 resin Thermal cell lysis EZNA kit.
Chelex-100 resin Thermal cell lysis CTAB enzymatic digestion (proteinase K)
chloroform EZNA kit.
Chelex-100 resin Thermal cell lysis CTAB enzymatic digestion (proteinase K and
RNase) by microwave EZNA kit.
Thermal cell lysis enzymatic digestion (proteinase K, lyticase and RNase) Chelex-100
resin EZNA kit

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A. Rodrguez et al. / Food Control 25 (2012) 666e672

instructions of the commercial DNA extraction kit AllPrep DNA/

RNA/Protein Mini Kit (Qiagen, Madrid, Spain).
2.3.4. Thermal breakage of conidia, CTAB extraction buffer and
EZNA kit (CTAB-EZNA method)
In this method, pellets were resuspended in 100 mL of sterile
nanopure water, boiled at 95  C for 10 min and cooled on ice for
10 min. Next, 500 mL CTAB lysis buffer (5 g D-sorbitol, 2 g N-lauroylsarcosine, 1.6 g/L CTAB, 1.4 M NaCl, 20 mM Na2EDTA, 2 g PVPP,
0.1 M TriseHCl, pH 8.0) buffer was added together with 10 mL of
a proteinase K solution (10 mg/mL) before incubation at 65  C for
1 h. In addition, 5 mL of 2-mercaptoethanol (SigmaeAldrich) was
included in the incubation at 65  C for 1 h (only in option 4b).
Samples were centrifuged at 13,000 rpm for 5 min, and the
supernatant was transferred to a new tube with 500 mL chloroform,
vortexed, and centrifuged at 13,000 rpm for 20 min. The upper layer
was transferred to a new tube and 10 mL RNase solution (10 mg/mL)
was added before incubation at 37  C for 1 h. An equal volume of
chloroform was then added, vortexed, and centrifuged at
13,000 rpm for 5 min. Finally, the aqueous phase was processed
according to instructions of the commercial DNA extraction of EZNA
Fungal DNA Mini Kit protocol (Omega bio-teck), starting from DNA
precipitation by adding 500 mL of isopropanol (step 4 protocol B).
2.3.5. Thermal breakage of conidia, Chelex-100 resin and EZNA kit
(Chelex100-EZNA method)
In this method, Chelex-100 resin (1 mg/10 mL, Sigma Aldrich) was
added to the ltrate obtained from spiked samples, and the sample
was boiled at 95  C for 10 min. An aliquot (1 mL) of each sample was
centrifuged at 12,000 rpm for 5 min. After centrifugation, supernatant was processed in three different ways: (a) according to instructions of the EZNA Fungal DNA Mini Kit protocol, starting from
applying sample to HiBind DNA column (step 10 protocol B); (b) by
mixing with 500 mL CTAB buffer and 10 mL of proteinase K (10 mg/mL)
before incubation at 65  C for 15 min. Next, 500 mL of chloroform was
added and centrifuged at 13,000 rpm for 5 min. Finally, supernatant
was also applied to HiBind DNA column (EZNA Fungal DNA Mini
Kit); (c) by mixing with 500 mL CTAB buffer and 15 mL of proteinase K
(10 mg/mL) before being microwaved at 625 W for 10 s. Next, 2 mL of
RNase solution (10 mg/mL) was added to the suspension and this was
microwaved again at 625 W for 10 s. Supernatant was then applied to
HiBind DNA column (EZNA Fungal DNA Mini Kit).
2.3.6. Thermal breakage of conidia, enzymatic treatment, Chelex100 resin and EZNA kit (Chelex100-enzymatic-EZNA method)
In this method the ltrate obtained from spiked Iberian drycured ham was boiled at 95  C for 10 min. Then, 50 mL of
proteinase K solution (10 mg/mL) was added together with 40 mL of
lyticase solution (10 mg/mL) before incubation at 65  C for 15 min.
Next, 20 mL of RNase solution (10 mg/mL) was added before incubation at 37  C for 15 min. Chelex-100 resin (1 mg/10 mL) was added
to the treated ltrate before incubation at 40  C for 5 min with
shaking (200 rpm). An aliquot (1 mL) of each sample was centrifuged
at 12,000 rpm for 5 min and supernatant was processed according to
the instructions of EZNA Fungal DNA Mini Kit protocol, starting
from applying sample to HiBind DNA column (step 10 protocol B).
In all of the above methods, the DNA obtained was eluted in
100 mL of elution buffer (provided by corresponding commercial
DNA extraction kits) and kept at 20  C until used as template for
PCR amplication.

above methods to estimate quality and quantity of fungal DNA. For

this, the primers Bt2a (50 -GGTAACCAAATCGGTGCTGCTTTC-30 ) and
Bt2b (50 -ACCCTCAGTGTAGTGACCCTTGGC-30 ) targeting the above
gene and previously used in a conventional PCR described by Glass
and Donalson (1995) was applied on genomic DNA extracted from
Iberian dry-cured ham inoculated with an OTA-producing strain
(P. nordicum CBS 110769) using the different DNA extraction
protocols. The reactions were performed in a programmable
thermal cycler, Mastercycler Gradient (Eppendorf, Hamburg, Germany) in volumes of 50 mL containing 4 mL of template DNA, 4 mL
each primer (10 mM), 5 mL of 10X PCR buffer, 2 mL of MgCl2 (50 mM),
0.4 mL of dNTPs (100 mM) and 0.5 mL of Taq DNA polymerase (2 U/
mL, Finnzymes, Espoo, Finland). The amplication program used
was: 1 cycle of 5 min at 94  C, 32 cycles of 1 min at 94  C, 1 min at
58  C and 1 min at 72  C and nally 1 cycle of 5 min at 72  C.
PCR product obtained was analyzed by electrophoresis in 2.0%
agarose gels. These gels were stained with ethidium bromide and
visualized with UV transillumination. Each amplication was done
in duplicate.
Quality and quantity of fungal DNA obtained by different DNA
extraction protocols was also tested by a specic SYBR Green qPCR
method based on the b-tubulin gene. Specic primers TubF1 (50 TCCCTTCGGCAAGCTTTTC-30 ) and TubR1 (50 -TGTTACCAGCACCGGACTGA-30 ) were designed to target a conserved region of the btubulin gene using Primer Express software (Applied Biosystems,
Foster City, CA, USA). This primer pair (TubF1/TubR1) amplied an
amplicon of 62 bp from nucleotide 426 to 488 bp according to the
published sequence of the b-tubulin gene (accession number
FN185734).
The qPCR analyses were performed on an Applied Biosystems
7500 Fast Real-Time PCR system (Applied Biosystems). qPCR reactions were prepared in MicroAmp optical 96-well reaction plates
and sealed with optical adhesive covers (Applied Biosystems). The
SYBR Green system with the designed primers was used. This qPCR
protocol was carried out in volumes of 25 mL containing 5 mL of
template DNA, 12.5 mL of 2x SYBR Premix Ex Taq (Takara Bio Inc.,
Otsu, Shiga, Japan), 0.5 mL of 50x ROX Reference Dye (Takara) and
1.5 mL of each primer (10 mM). The amplication program used was:
1 cycle of 10 min at 95  C and 40 cycles 95  C for 15 s and 60  C for
1 min. After the nal PCR cycle, melting curve analysis of the PCR
products was performed by heating to 60e95  C and continuous
measurement of the uorescence to verify the PCR product. All PCR
reactions were performed in duplicates and control sample without
DNA template were included in all runs. Threshold cycle (Ct) value,
corresponding to the PCR cycle number at which uorescence was
detected above threshold, was calculated by the 7500 Fast System
SDS software (Applied Biosystems). The recovery (R) was calculated
according to Jara et al. (2008):

%R


 
.
NTC  Ctminimal  Ctsample  Ctminimal


NTC  Ctminimal  100;

where Ctminimal is the lowest Ct value obtained from DNA extracted

from the same solution of pure conidia used for spiking food
samples at the nal concentration of 2.5  103 spores/ml and the
Ctsample is Ct value obtained from DNA extracted from every spiked
food sample. NTC is the Ct value for the non-template control.
2.5. Evaluation of DNA extraction protocols for quantication of
OTA producers

2.4. PCR assays to detect and quantify b-tubulin gene

Conventional and qPCR assays designed to detect the universal
fungal b-tubulin gene were assayed with the DNA obtained from the

DNA obtained from the Penicillium producer with the different

extraction methods was evaluated by a specic SYBR Green qPCR
method to detect and quantify OTA-producing molds. Two specic

A. Rodrguez et al. / Food Control 25 (2012) 666e672

primers, F-npstr (50 -GCCGCCCTCTGTCATTCCAAG-30 ) and R-npstr

(50 -GCCATCTCCAAACTCAAGCGTG-30 ) were designed based on nonribosomal peptide synthetase (otanpsPN) gene (accession number
AY557343). The above primers amplied a PCR product of 117 bp.
This qPCR protocol was carried out in a nal volume of 25 mL,
containing 5 mL of template DNA, 12.5 mL of 2x SYBR Premix Ex Taq
(Takara), 0.5 mL of 50x ROX Reference Dye (Takara) and 400 nM of
both F-npstr and R-npstr primers. The qPCR was conducted by the
thermal cycling conditions described in 2.4 section. All PCR reactions were performed in duplicates and control sample without
DNA template were included in all runs. The Ct value was calculated
as the cycle number at which the concentration increase became
exponential. DNA recovery was obtained as indicated in Section 2.4.
2.6. Selection and validation of DNA extraction protocol
The DNA extraction method, which showed amplication with
the conventional PCR for the b-tubulin gene and the highest DNA
recovery values in the qPCR assays for the b-tubulin and otanpsPN
genes, was selected for further validation in different food matrices.
To validate the selected method, ve different foods (Iberian
dry-cured ham, ripened cheese, peanuts, pistachios and grapes)
and two OTA-producing strains of different genus, (P. nordicum CBS
110769 and A. westerdijkiae CECT 2948) were used.
For this, the above foods were separately inoculated with spores
of the two former reference strains at level of 2.5  103 spores/g.
DNA extraction was carried out following the most efcient method.
qPCR reactions to quantify b-tubulin and otanpsPN genes and to
estimate the DNA recovery were carried out as above described in
Section 2.4 and 2.5, respectively, using 5 mL of DNA in duplicate.
2.7. Statistical analysis
All the statistical analyses were performed with the SPSS v.15.0.
One way analysis of variance (ANOVA) was carried out to determinate signicant differences within and between groups. Tukeys
test was applied to compare the mean values. Statistical signicance was set at P  0.05.
3. Results

669

Bt2a/Bt2b primers and a conventional PCR system, an amplicon of

453 bp was only found with DNA obtained from EZNA, QIAGEN, CTABEZNA (option 4a), Chelex100-EZNA (option 5a) and Chelex100enzymatic-EZNA methods. With the TubF1/TubR1 primer pair and
a qPCR assay a specic product of 65 bp was amplied with a Tm
value of 82.2  0.3  C from all DNA extracted from the different
protocols, except for CTAB-EZNA (option 4b) and Chelex100-EZNA
(option 5c) methods. The DNA relative recoveries estimated by
SYBR Green qPCR based on b-tubulin gene ranged from 17.9 to 99.3%,
but most of the assayed methods showed recoveries higher than
70%. Maximum extraction was obtained with Chelex100-enzymaticEZNA, CTAB-EZNA (option 4a) and Chelex100-EZNA (option 5a)
methods. To evaluate the DNA extraction methods in the use for
quantication of OTA-producing molds by qPCR, a specic SYBR
Green method based on otanpsPN gene was used. With the F-npstr/
R-npstr primers a PCR product of the expected size of 117 bp showing
a Tm value of 84.6  0.3  C was obtained for all DNA obtained with
the different extraction protocols tested. The DNA relative recoveries
ranged from 28.8 to 95.5%. Maximum recovery was achieved with
Chelex100-enzymatic-EZNA and CTAB-EZNA (option 4a) methods.
3.2. Validation of the Chelex100-enzymatic-EZNA DNA extraction
protocol
The Chelex100-enzymatic-EZNA method (Fig. 1) was selected for
further validation assays on different food matrices, since DNA obtained
with this method showed amplication by conventional PCR for btubulin gene and yielded the maximum DNA recoveries by both qPCR
based on b-tubulin and otanpsPN genes. The DNA relative recoveries
obtained with this method in the different food matrices tested by qPCR
based on b-tubulin gene ranged from 61 to 99 % (Table 3). The recoveries
obtained from different matrices spiked with P. nordicum and
A. westerdijkiae were more than 73 and 61%, respectively. In general, the
DNA recoveries from P. nordicum were higher in all matrices than those
obtained for A. westerdijkiae, except when grape was the food matrix.
Similar results were obtained when DNA recovery was estimated by the
qPCR assay for the otanpsPN gene. All DNA recoveries were higher than
69% for all food matrices tested (Table 3) and recoveries were in general
higher for P. nordicum than for A. westerdijkiae. Furthermore, the DNA
recoveries obtained were higher than 90% in 3 of 5 foods tested (Iberian
dry-cured ham, ripened cheese and peanut).

3.1. Evaluation of DNA extraction methods

4. Discussion
To test the quality of the obtained DNA and the absence of PCR
inhibitors a conventional PCR and an SYBR Green qPCR system based
on amplication of the b-tubulin gene were used (Table 2). With

The correct quantication of OTA-producing molds in foods is

clearly inuenced by the DNA extraction method. In the present

Table 2
Amplication by conventional PCR based on b-tubulin and DNA recoveries calculated from qPCR based upon b-tubulin and otanpsPN genes to validate the DNA quality obtained
using the six extraction methods from Iberian dry-cured ham inoculated with P. nordicum CBS 110769.
DNA extraction methods

1.Mechanical-EZNA
2. EZNA
3. QIAGEN
4. CTAB-EZNA

2a (isopropanol precipitation)
2b (isopropanol:ethanol precipitation)

4a (without 2-mercaptoethanol)
4b (with 2-mercaptoethanol)
5.Chelex100-EZNA
5a (EZNA kit)
5b (CTAB and EZNA kit)
5c (CTAB, microwave and EZNA kit)
6.Chelex100-enzymatic-EZNA
a
b
c
d
e

Amplication of b-tubulin gene

Amplication of otanpsPN gene

Conventional PCR

Recovery determined by qPCR

Recovery determined by qPCR

c,d

67.5
72.7
61.4
68.4
88.7
43.6
76.7
28.8
31.7
95.5




84.4
90.3
86.3
73.3
98.3
e
nd
94.9
17.9
nd
99.3







0.11
0.07
0.46
0.41
0.30

 0.66
 0.44
 0.25

Indicates which the amplicon of 453 bp was not observed.

Indicates which the amplicon of 453 bp was observed.
DNA recovery was calculated by %R ((NTC e Ctminimal) e (Ctsample e Ctminimal))/(NTC e Ctminimal))  100.
Results obtained in duplicate.
nd: amplication was not detected.












0.32
015
0.08
0.57
0.39
0.32
0.90
0.10
0.18
0.89

670

A. Rodrguez et al. / Food Control 25 (2012) 666e672

5 g spiked food sample + 10 mL TrisHCl buffer

Homogenization

Filtrate (10 mL)

Boiling at 95 C for 10 min

Adition of proteinase K and lyticase

before incubation at 65 C for 15 min

Adition of RNAse before incubation at

37 C for 15 min

Adition of Chelex-100 resin (1 mg)

before incubation at 40 C for 5 min
(200 rpm)
1 mL of Chelex-treated
sample

Centrifugation at 12,000 rpm for 5 min

Supernanant

EZNA Fungal DNA Mini Kit (step 10 protocol B)

PCR assays
Fig. 1. Schematic diagram showing the selected procedure (Chelex100-enzymatic-EZNA
method) for the extraction of genomic DNA from OTA-producing molds in foods.

study, six different DNA extraction methods for foods were evaluated to obtain fungal DNA of high quality and without PCR inhibitors to be used for quantication of OTA-producing molds by qPCR.
Initially, the quality of the DNA and the absence of inhibitors were
tested using the amplication of the b-tubulin gene by conventional
and qPCR methods. The b-tubulin gene is a highly conserved gene

that is ubiquitous in all eukaryotic cells (Cabaas, Castell, Abarca,

Bragulat, & Cabaes, 2009), and it has previously been applied in
PCR methods used for testing for the presence of fungal DNA (Atoui
et al., 2007). The results obtained with the conventional PCR based
on b-tubulin gene agree with those obtained by qPCR for this gene.
Thus, when there was no amplication with conventional PCR,
amplication was not detected or low DNA recovery was obtained
by qPCR. The only exception was observed with DNA extracted from
Mechanical-EZNA method that did not yield amplicon by conventional PCR and showed a DNA recovery of 84% in the qPCR. This
could be due to the lower sensitivity of the conventional PCR as
compared to the qPCR method which generally detects lower
amount of DNA than conventional PCR tests (Jara et al., 2008).
Comparison of the different methods based on the results of the
qPCR targeting the b-tubulin gene showed that the maximum DNA
recoveries were obtained with Chelex100-enzymatic-EZNA, CTABEZNA (option 4a) and Chelex100-EZNA (option 5a). Thus, the presence of Chelex resin, a commercially available polystyrenedivinylbenzene iminodiacetate material that chelates heavy
metals which inhibit enzymatic activity in PCR could improve the
DNA recovery as has been reported by Ishii and Loynachan (2004).
Use of Chelex-100 resin prior to cell lysis has previously been
described in a method for quantication of Listeria monocytogenes
in fruit juice (Kim & Cho, 2010) but has never been described for
mycotoxigenic molds.
Furthermore, from the results it can deduced that the effect of
Chelex-100 in DNA extraction depends on the method it is
combined with. Thus, the combination of Chelex-100 with
a commercial kit (Chelex100-EZNA method, option 5a) or/and an
adequate
enzymatic
treatment
(Chelex100-enzymatic-EZNA
method) after thermal disruption of conidia contribute to an efcient DNA extraction, since DNA obtained with these methods did
not show sign of inhibition in conventional PCR and gave high DNA
recoveries (94.9 and 99.3%, respectively). In addition, in the case of
the Chelex100-enzymatic-EZNA method, the enzymatic digestion
with proteinase K and lyticase contribute to an efcient DNA yield
(Snchez, Rodrguez, Casado, Martn, & Crdoba, 2008). Lyticase is
an enzyme which degrades fungal cell walls by cleaving the b(1e3)
glycosidic bonds between glucose moieties resulting in the breakdown of the rigid, water insoluble skeletal portion of the cell wall
(McDevitt, Lees, Merz, & Schwab, 2004), being able to improve the
DNA extraction. The combination of Chelex-100 and enzymatic
treatment has been reported previously in development of PCR
tests (Garca Gonzlez, Rodrigo Tapia, Snchez Lazo, Ramos, &
Surez Nieto, 2004; Reyes-Escogido et al., 2010), but never in the
extraction of fungal DNA suitable for qPCR.
The combination of commercial kits with previously described
treatments of food samples (specic lysis buffers, enzyme digestion
or thermal disruption) improved yield and quality of the extracted
fungal DNA. Thermal disruption of spores, digestion with
proteinase K in combination with a CTAB buffer and a commercial

Table 3
DNA recoveries calculated from qPCR based upon b-tubulin and otanpsPN genes to validate the DNA quality obtained using the Chelex100-enzymatic-EZNA method considering
the matrix in both OTA-producing molds.
Foods

Amplication of b-tubulin gene

Amplication of otanpsPN gene

Recovery determined by qPCR

Iberian dry-cured ham

Ripened cheese
Peanut
Pistachio
Grape
a
b

Recovery determined by qPCR

Penicillium nordicum

Aspergillus westerdijkiae

a,b

71.9
74.4
61.3
79.6
82.3

99.3
80.7
73.5
94.9
77.4







0.25
0.18
0.17
0.25
0.35







0.94
0.38
0.02
0.41
0.18

DNA recovery was calculated by %R ((NTC  Ctminimal) e (Ctsample e Ctminimal))/(NTC e Ctminimal))  100.
Results obtained in duplicate.

Penicillium nordicum
95.5
92.1
75.5
99.5
72.0







0.89
0.47
0.28
0.30
0.37

Aspergillus westerdijkiae
79.9
89.6
74.1
69.6
78.6







0.39
0.52
0.94
0.13
0.82

A. Rodrguez et al. / Food Control 25 (2012) 666e672

extraction kit for fungal DNA (CTAB-EZNA method option 4a)

showed no sign of inhibition of conventional PCR and a good
recovery was obtained (over 98%). CTAB is a cationic surfactant that
is added to solubilize the membranes (Alame & Jrviste, 1995),
which helps to remove capsular polysaccharides (Spitzer & Spitzer,
1992) and precipitate the DNA (Sibatani, 1970). However, the
addition of 2-mercaptoethanol to the CTAB extraction buffer (CTABEZNA method option 4b) reduced the DNA yield. This fact could be
due to the above organic solvent is a strong reducing reagent which
can act as a pro-oxidant during nucleic acid extraction inhibiting
the action of RNase in the DNA extraction procedure (Hofer, Seo,
Prudencio, & Leeuwenburgh, 2006).
To test the use of the extracted DNA in the quantication of OTAproducing molds in food, a primer pair designed from the otanpsPN
gene (accession number AY557343) which was characterized by
Geisen, Schmidt-Heydt, and Karolewiez (2006) and belongs to
a gene cluster of the OTA biosynthethic pathway in Penicillium was
used. The results obtained with the qPCR assay for otanpsPN were in
accordance with those obtained by amplication of b-tubulin gene
by qPCR. The highest recovery values were obtained with CTABEZNA (option 4a) and Chelex100-enzymatic-EZNA methods (89 and
96%, respectively). In addition, the last method could be performed
within a short time period (1 h) instead of 5 h needed for performing the CTAB-EZNA method. Ideally, a DNA extraction method
should be simple, quick and efcient. Thus, the Chelex100enzymatic-EZNA method was selected to quantify OTA-producing
molds in spiked foods due to its optimal trade-off between DNA
extraction time needed, cost (material and labor), the optimal yield
of DNA and the absence of substances that could inuence the PCR
reaction.
To validate the selected method, DNA was extracted from 5
different foods inoculated with 2 OTA-producing species. The
results obtained by qPCR on b-tubulin gene showed differences
between the DNA relative recoveries ranging from 73 to 99% for
P. nordicum and from 61 to 82% for A. westerdijkiae. When testing
the extracted DNA with the assay for OTA-producing molds the
mean relative recoveries for both species in all matrices ranged
from 69 to 99%, again showing higher recoveries from P. nordicum
than for A. westerdijkiae. This difference between the two species
may be due to the different surface characteristics of Penicillium
spores as compared to Aspergillus spores (Fisher & Richmond, 1970;
Hess & Stocks, 1969), which may make DNA extraction slightly
easier in Penicillium spp. The type of food matrix have some effect
on the DNA recovery, although in all cases the obtained values are
higher than those reported (60%) as acceptable to quantify DNA by
qPCR (Jara et al., 2008). The lowest values observed in peanut and
grape could be due to the high concentration of peanut oils and
polyphenols present in grapes which can affect qPCR (Passone
et al., 2010; Selma et al., 2008).
The present study showed that the quality of DNA extracted
with Chelex100-enzymatic-EZNA protocol was matrix and species
dependent, which is in accordance with other similar studies (Jara
et al., 2008). However, all DNA recoveries were above 69% and
therefore acceptable for use in routine analysis to quantify OTAproducing molds by qPCR in foods. Only for complex matrices
such as formulated foods, an intensive extraction method including
a lysis extraction buffer such as the CTAB-EZNA method could be
useful since this method gave DNA recoveries similar to the more
rapid Chelex100-enzymatic-EZNA protocol.
In conclusion, the method based on thermal disruption of conidia, enzymatic treatment of samples and use of Chelex-100 resin
(Chelex100-enzymatic-EZNA method) could be considered for
routine analysis in HACCP systems in the food industry to obtain
good yield of DNA from foods for quantifying OTA-producing molds
by qPCR.

671

Acknowledgments
This work has been funded by project AGL2007-64639 of the
Spanish Comision Interministerial de Ciencia y Tecnologa, Carnisenusa CSD2007-00016, Consolider Ingenio 2010 and GRU08100
and GRU09158 of the Junta de Extremadura and FEDER. Alicia
Rodrguez would like to thank the Spanish Comision Interministerial de Ciencia y Tecnologa for the pre-doctoral grant (BES-2008008021). The work of Annemarie F. Justesen was supported by The
Danish Food Industry Agency, project 3412-07-01873.
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