Quantification of Ochratoxin A–Producing Fungi in Coffee Products Using Quantitative PCR

Abstract

Ochratoxin A (OTA) is a polyketide mycotoxin that is produced by Aspergillus and Penicillium. Food contaminated with OTA poses health risks and is a food-safety challenge. Quantitative polymerase chain reaction (qPCR) has been used to identify non-toxigenic and toxigenic strains from coffee samples using polyketide synthase (pks), the OTA synthesis gene. In this research, Aspergillus carbonarius (ochratoxin-producing strain) and A. flavus (non-ochratoxin-producing strain) were used to amplify a 141 bp fragment of the pks gene. The 141 bp PCR product was successfully cloned into TOPO®TA plasmid. Subsequently, ten-fold dilutions of plasmid DNA were used to generate the standard curve by plotting the threshold cycle against log DNA concentration using qPCR. Further, fungal DNA contamination was quantified in 11 samples of roasted coffee using qPCR. All 11 coffee samples were accepted as safe, since the fungal genomic DNA contamination was less than 3.85 x 103 copies. Therefore, this research suggested that qPCR is a fast and accurate method to detect and quantify OTA-producing fungi in coffee products. Thus, we successfully developed a system to quantify fungal contamination in coffee.

Full text

CMU J. Nat. Sci. (2018) Vol. 17(1) 39
Quantication of Ochratoxin A–Producing Fungi
in Coee Products Using Quantitative PCR
Jakapan Potipun, Wijittra Ruaylarb, Rungrote Nilthong and
Amorn Owatworakit*
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
*Corresponding author. E-mail: amorn@mfu.ac.th
https://doi.org/10.12982/CMUJNS.2018.0004
ABSTRACT
Ochratoxin A (OTA) is a polyketide mycotoxin that is produced by Aspergillus and
Penicillium. Food contaminated with OTA poses health risks and is a food-safety challenge.
Quantitative polymerase chain reaction (qPCR) has been used to identify non-toxigenic
and toxigenic strains from coee samples using polyketide synthase (pks), the OTA
synthesis gene. In this research, Aspergillus carbonarius (ochratoxin-producing strain)
and A. avus (non-ochratoxin-producing strain) were used to amplify a 141 bp fragment
of the pks gene. The 141 bp PCR product was successfully cloned into TOPO®TA plasmid.
Subsequently, ten-fold dilutions of plasmid DNA were used to generate the standard curve
by plotting the threshold cycle against log DNA concentration using qPCR. Further, fungal
DNA contamination was quantied in 11 samples of roasted coee using qPCR. All 11
coee samples were accepted as safe, since the fungal genomic DNA contamination was
less than 3.85 x 103 copies. Therefore, this research suggested that qPCR is a fast and
accurate method to detect and quantify OTA-producing fungi in coee products. Thus, we
successfully developed a system to quantify fungal contamination in coee.
Keywords: Aspergillus carbonarius, Ochratoxin A, Coee, Quantitative PCR, Polyketide
synthase (pks) gene
INTRODUCTION
Ochratoxin A (OTA) is a pentaketide mycotoxin that exhibits immunosuppressive,
teratogenic, and carcinogenic properties. OTA is also a potent nephrotoxin and the possible
causative agent of Balkan endemic nephropathy in humans (Leong et al., 2006). The toxin can
be found in a broad range of processed and unprocessed foodstus, including coee.
Coee is one of the most popular drinks in the world and a valuable primary product.
The presence of OTA in the various stages of coee processing is of great concern. The major
source of OTA in coee is fungi of the genus Aspergillus (Pardo et al., 2004; Velmourougane
et al., 2011). Specically, Aspergillus carbonarius is one of the main species responsible for
production and accumulation of the toxin in coee. This fungus has also been reported to have
the highest ochratoxigenic potential (Mulè et al., 2006).

CMU J. Nat. Sci. (2018) Vol. 17(1)40
Given the negative eects of OTA, it is important to have a reliable method for
detecting and quantifying OTA-producing fungi in foodstus. Conventional techniques are
not accurate. Moreover, identifying Aspergillus species based on morphological characters is
dicult and requires taxonomic expertise. Furthermore, spore isolation and enumeration may
introduce bias against slow-growing species (Selma et al., 2008). Previous studies have shown
that detecting OTA-producing Aspergillus using PCR methods is specic, sensitive, rapid, and
easy to automate. (Atoui et al., 2007).
Specically, quantitative polymerase chain reaction (qPCR) is a powerful tool that
combines uorescent dyes and sequence-specic primers to monitor accumulation of PCR
product during the procedure. Furthermore, it does not require other post-amplication
procedures, such as gel electrophoresis. Thus, this technique is highly reliable, sensitive, and
suitable for high throughput analysis (Mulè et al., 2006). Quantitative PCR has been used to
quantify OTA-producing fungi in many agricultural products, including wine, cereal grain,
tea, and coee (Atoui et al., 2007). Common targets include polyketide synthase (pks), a gene
involved in synthesis of secondary metabolites and OTA, as well as internal transcribed spacer
(ITS) rRNA (Sartori et al., 2006; Atoui et al., 2007).
This work aimed to develop a method for quantifying A. carbonarious, an OTA-
producing fungus, in coee products using qPCR to provide a useful food-safety tool.
MATERIALS AND METHODS
Coee samples and fungal strains
Eleven roasted coee products were purchased from coee companies in Chiang Rai
province. A. carbonarius TISTR3214 (OTA-producing strain) and A. avus TISTR3130
(non-OTA–producing strain) were obtained from the Thailand Institute of Scientic and
Technological Research (TISTR) culture collection and used as reference strains. Fungal
strains were grown at 30°C on potato dextrose agar for 7 days. Subsequently, their spores
were collected in 0.1% (v/v) between 80 and cultivated in 100 ml of potato dextrose broth at
30°C without shaking for 3 days.
Fungal genomic DNA extraction from reference strains
One hundred mg of fungal mycelia from A. carbonarius were frozen in liquid nitrogen
before nucleic acid extraction. Genomic DNA was extracted using the DNA secure Plant Kit
(TIANGEN) following manufacturer’s instructions. The quality and quantity of DNA were
estimated by using the OD260/280 ratio and 3% agarose gel electrophoresis.
Fungal DNA extraction from coee samples
Five g of roasted coee beans were soaked in 10 mL lysis buer (66mM EDTA, 33mM
Tris, 3.3% Triton X-100, 1.65M Guanidinium-HCl, 0.825M NaCl, 6% Polyvinyl pyrrolidone-
40T, pH=7.9), added to 10 mL ddH2O, and then shaken vigorously by hand for 1 min. One
mL of supernatant was taken and mixed with 0.4 mL of absolute ethanol. DNA was extracted
using the Tiangen® DNA extraction kit. Subsequently, DNA solutions were kept at - 20°C.

CMU J. Nat. Sci. (2018) Vol. 17(1) 41
PCR reaction
The total volume of the PCR reaction was 20 µl reaction, which contained: 2 µl of Taq
polymerase buer 10x, 2 µl of dNTP, 1µl of each primer, 1 µl of Taq, about 30 ng of genomic
DNA, topped up to 20 µl with dH2O. Reaction conditions were: 94°C for 4 min; followed by
28 cycles of 94°C for 40s, 65°C for 40s, and 72°C for 40s; and nal extension at 72°C for 10
min. The primer pair, OTAF (5’-AATATATCGACTATCTGGACGAG CG-3’) and OTAR (5’-
GAAGCCCTCTGCGATCTCCC-3’) was used to amplify a 141 bp fragment of the pks gene.
The amplied products were examined by 3% agarose gel electrophoresis (Atoui et al., 2007).
Cloning of pks gene and analysis
The TOPO TA Cloning® Kit (Invitrogen) was used for cloning. The PCR products were
inserted into the vector and the bacterial E. coli DH5α cells were transformed using the heat
shock method. Subsequently, the recombinant cells were cultured on Luria-Bertani medium
for 24 hr. Positive colonies were picked and plasmid isolation was performed using TIANprep
Rapid Mini Plasmid Kit® (TIANGEN).
DNA sequencing analysis
The extracted plasmids were bidirectionally sequenced using OTA primers (First BASE
Laboratories, Malaysia).
Real-time qPCR reaction
Quantitative PCR reactions were run in triplicate. Each reaction well contained 10 μl
of template DNA, 5 μl of SYBRR® Green I, and 0.5 µL of each forward and reverse OTA
primer. Real-time qPCR was conducted using the following cycling conditions: 50°C for 2
min, 95°C for 10 min, followed by 40 cycles of 95°C for 15s, and 60°C for 60s. The DNA
standard curve was generated by plotting the threshold cycle (Ct) versus the logarithm of
known DNA concentrations using a series of 10-fold dilutions of a plasmid containing the pks
gene (ranging from 3x108-3x103 DNA copies). Quantication of A. carbonarius pks gene was
performed by running the DNA from the coee samples in parallel with the serial dilution
standard.
RESULTS
Amplication of pks gene from fungal strains
A fragment of the pks gene was amplied only from A. carbonarious using specic
OTA primers. The PCR product was of the expected size of 141 bp (Figure 1). A. avus, the
non OTA-producing fungus gave a negative result. These results showed that the primers used
for PCR amplication were specic for targeting pks gene in A. carbonarious.

CMU J. Nat. Sci. (2018) Vol. 17(1)42
Figure 1. 3% agrose gel electrophoresis of PCR products from the fungal strains amplied
with OTA primers. Lane M contains the 25 bp DNA ladder; Lanes 1 and 2 contain
the pks gene of A. carbonarious; Lanes 3 and 4 are A. avus.
OTA cloning and sequencing analysis
A 141 bp fragment of the pks gene from extracted plasmids was amplied using PCR
(Figure 2.). The obtained pks gene sequences were then compared with the database in the
National Center for Biotechnology and Information (NCBI) using the BLAST search tool.
The fragment was conrmed as pks based on 98% identity with the already published A.
carbonarius pks sequence (Atoui et al., 2006).
Figure 2. 3% agrose gel electrophoresis of the 141 bp pks fragment amplied with OTA
primers. Lane M contains the 25 bp DNA ladder; Lanes 1, 2, and 3 contain the
TOPO plasmid with the 141 bp fragment of pks gene.
Amplication of OTA gene from coee samples
All 11 coee samples were positive for the pks gene as indicated by the presence of the
specic PCR product amplied with OTA primers (Figure 3). This result demonstrated fungal
contamination in commercially available coee in Chiang Rai.
Figure 3. 3% agrose gel electrophoresis of the 141 bp fragment of pks gene amplied with
OTA primers. Lane M contains the 25 bp DNA ladder; Lanes 1-11 show the PCR
products obtained from coee samples.
Quantication of A. carbonarious using qPCR
For every qPCR run, a standard curve was constructed using triplicate reactions of
10-fold dilutions of A. carbonarius plasmid DNA ranging from 3x103-3x108 DNA copies
conjugated with SYBR® Green I dye. The standard curve was obtained by plotting the
threshold cycle (Ct) corresponding to the logarithm of the plasmid DNA concentration of

CMU J. Nat. Sci. (2018) Vol. 17(1) 43
each dilution (Table 1). The linear correlation coecient of the standard curve was R2=0.99,
and melt curve analysis indicated the specicity and accuracy of the PCR-based quantication
(Figure 4).
The Ct values of the 11 coee samples obtained from the real-time PCR system ranged
from 24 to 31 (Table 2), indicating that only a small amount of fungal DNA was present in
the coee samples used in this experiment. Only coee sample no. 8 could be quantied
containing 3.85x103 DNA copies. The rest of the samples could not be reliably estimated
using the equation from the standard curve (Figure 4B). This indicated that the DNA copy
number of OTA-producing fungi must be less than 3.85x103. Consistently, the total number
of fungal species in roasted coee using culture-based approaches was 10 and 5x103 CFU/5g
(data not shown).
Table 1. Ct values corresponding to the standard curve obtained using SYBR-Green I with
genomic DNA from A. carbonarius.
Ten-fold dilutions DNA copy equivalents Threshold cycle (Ct) ± SD
Dilution 1 3 x 108 7.88± 0.10
Dilution 2 3 x 10711.20± 0.03
Dilution 3 3 x 10614.87± 0.12
Dilution 4 3 x 10518.40± 0.18
Dilution 5 3 x 10421.86± 0.29
Dilution 6 3 x 10325.36± 0.16
Figure 4. (A) A plot of relative uorescence units corresponding to cycle number of the
amplication. (B) Standard curve showing the log10 DNA amount (ng) vs.
threshold cycle of the real-time PCR method for the 10-fold serial dilutions of
A. carbonarius.

CMU J. Nat. Sci. (2018) Vol. 17(1)44
Table 2. Threshold cycle (Ct) value of 11 coee samples obtained from real-time PCR.
Sample number Threshold cycle (Ct) ± SD DNA copy equivalents (copies)*
S01 28.20±0.20 3.6x10
S02 28.40±0.14 3.9x10
S03 28.68±0.76 2.6x10
S04 28.94±0.07 <10
S05 29.22±0.07 <10
S06 28.75±0.20 2.5x10
S07 28.22±0.57 3.5x10
S08 24.57±0.15 3.85x103
S09 27.65±0.21 5.1x10
S10 27.51±0.20 5.6x10
S11 31.50±0.21 <10
Note: The values were derived from averaging three replicates. *The pks gene copy number equivalents of A.
carbonarius from coee samples were calculated using a standard equation.
DISCUSSION
Aspergillus species produce ochratoxin A, which is a toxic secondary metabolite. This
mycotoxin is nephrotoxic and carcinogenic and has been detected in cereal and other food
commodities, such as coee (Bucheli et al., 1998). The PCR reaction has been used to detect
and quantify mycotoxin-producing Aspergillus species. In this study, we used specic OTA
primers to detect pks, the OTA synthesis gene, in fungal strains (Figure 1). Previous research
has shown that amplication of pks gene in Aspergillus species using these primers was
specic to A. carbonarius only, as indicated by the specic 141 bp PCR product (Atoui et al.,
2007).
Specicity of the primer pair OTAF/OTAR was comrmed using PCR assay. The
resulting amplicon was then cloned into a plasmid. The sequence of the amplicon had a
98% identity to the published A. carbonarius polyketide synthase 5 (accession number:
HM026487) in GenBank (Atoui et al., 2006).
In order to examine the presence of OTA-producing fungi in the 11 DNA samples
extracted from coee, PCR reactions were performed using OTAF/OTAR primers to amplify
a 141 bp fragment of the pks gene. The pks gene was detected in all coee samples (Figure 3
and 4).
This study shows that qPCR is a useful tool for detecting and quantifying A. carbonarius
in coee samples or other food products. It is necessary to use rapid and specic methods
for early detection of OTA-producing fungi, especially when foods are involved (Dao et al.,
2005). Recently, a rapid, specic, and sensitive qPCR assay for detecting and quantifying A.
carbonarius on coee was developed. In our experiment, we generated the standard curve,

CMU J. Nat. Sci. (2018) Vol. 17(1) 45
which was obtained by plotting the Ct value versus the log of the concentration of each DNA
dilution (Figure 4) with the regression coecient (R2) greater than 0.99. The qPCR method
was highly sensitive; the smallest amount of fungal DNA that it detected was 3.85x103 DNA
copies. To test the ability of the qPCR method to quantify OTA-producing fungi in coee
samples, the Ct values of the samples (Table. 1) were compared with the standard curve.
Given the detection limits of real-time PCR systems, very low concentrations of DNA can
lead to diculty in detecting uorescence due to the low amount of SYBR intercalating
with the sample. The maximum Ct value for detection in our work was 25. Our data are in
agreement with previous results that also used qPCR assays for A. carbonarius quantication
in articially inoculated samples (Atoui et al., 2007). The A. carbonarius DNA concentration
was quantied in coee samples and was between 10-14-10-9g DNA/5g of coee sample.
CONCLUSION
This research successfully identied the pks gene of A. carbonarius in Arabica coee
by using specic OTAF/OTAR primers. We developed a real-time PCR to quantify fungal
DNA concentration in Arabica coee. In addition, this technique was specic, as well
as, sensitive. The described method could be a useful tool for screening, monitoring, and
detecting contamination with OTA-producing fungi in commercial food products. Further
study is required to quantify the concentration of OTA contamination in coee products.
ACKNOWLEDGEMENTS
This research was supported by a grant from Mae Fah Luang University and presented
at the 28th Annual Meeting of the Thai Society for Biotechnology and International Conference.
We also thank Dr. Khanobporn Tangtrakulwanich and Dr. Gentekaki Eleni for help with the
manuscript.
REFERENCES
Atoui, A., Dao, H.P., Mathieu, F., and Lebrihi, A. 2006. Amplication and diversity analysis of
ketosynthase domains of putative polyketide synthase genes in Aspergillus ochraceus
and Aspergillus carbonarius producers of ochratoxin A. Molecular Nutrition and Food
Research. 50(6): 488-493. https://doi.org/10.1002/mnfr.200500165
Atoui, A., Mathieu, F., and Lebrihi, A. 2007. Targeting a polyketide synthase gene for
Aspergillus carbonarius quantication and ochratoxin A assessment in grapes using
real-time PCR. International Journal of Food Microbiology. 115(3): 313-318. https://
doi.org/10.1016/j.ijfoodmicro.2006.11.006
Bucheli, P., Meyer, I., Pittet, A., Vuataz, G., and Viani, R. 1998. Industrial storage of green
robusta coee under tropical conditions and Its impact on raw material quality and
ochratoxin A content. Journal of Agricultural and Food Chemistry. 46(11): 4507-4511.
https://doi.org/10.1021/jf980468+

CMU J. Nat. Sci. (2018) Vol. 17(1)46
Dao, H.P., Mathieu, F., and Lebrihi, A. 2005. Two primer pairs to detect OTA producers by
PCR method. International Journal of Food Microbiology. 104(1): 61-67. https://doi.
org/10.1016/j.itfoodmicro.2005.02.004
Leong, S.L., Hocking, A.D., Pitt, J.I., Kazi, B.A., Emmett, R.W., and Scott, E.S. 2006.
Australian research on ochratoxigenic fungi and ochratoxin A. International Journal
of Food Microbiology. 111, Supplement 1(0): S10-S17. https://doi.org/10.1016/j.
itfoodmicro.2006.02.005
Mulè, G., Susca, A., Logrieco, A., Stea, G., and Visconti, A. 2006. Development of a
quantitative real-time PCR assay for the detection of Aspergillus carbonarius in
grapes. International Journal of Food Microbiology. 111(SUP.1): S28-S34. https://doi.
org/10.1016/j.ijfoodmicro.2006.03.010
Pardo, E., Marı́n, S., Solsona, A., Sanchis, V., and Ramos, A.J. 2004. Modeling of germination
and growth of ochratoxigenic isolates of Aspergillus ochraceus as aected by water
activity and temperature on a barley-based medium. Food Microbiology. 21(3): 267-
274. https://doi.org/10.1016/j.fm.2003.09.001
Selma, M.V., Martínez-Culebras, P.V., and Aznar, R. 2008. Real-time PCR based procedures
for detection and quantication of Aspergillus carbonarius in wine grapes. International
Journal of Food Microbiology. 122(1-2): 126-134. https://doi.org/10.1016/j.
ijfoodmicro.2007.11.049
Sartori, D., Furlaneto, M.C., Martins, M.K., Ferreira de Paula, M.R., Pizzirani-Kleiner, A.A.,
Taniwaki, M.H., and Fungaro, M.H.P. 2006. PCR method for the detection of potential
ochratoxin-producing Aspergillus species in coee beans. Research in Microbiology.
157(4): 350-354. https://doi.org/10.1016/j.resmic.2005.09.008
Velmourougane, K., Bhat, R., Gopinandhan, T.N., and Panneerselvam, P. 2011. Management
of Aspergillus ochraceus and Ochratoxin-A contamination in coee during on-farm
processing through commercial yeast inoculation. Biological Control. 57(3): 215-221.
https://doi.org/10.1016/j.biocontrol.2011.03.003