Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.

[1]  Alberto Martín,et al.  Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers. , 2008, Journal of food protection.

[2]  N. Lima,et al.  A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds. , 2009, International journal of food microbiology.

[3]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[4]  R. García-Villanova,et al.  Aflatoxins and ochratoxin A in red paprika for retail sale in Spain: occurrence and evaluation of a simultaneous analytical method. , 2008, Journal of agricultural and food chemistry.

[5]  M. Ahmedna,et al.  Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. , 2005 .

[6]  C. Monnet,et al.  Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR , 2006, Applied and Environmental Microbiology.

[7]  C. Vázquez,et al.  ITS-based detection and quantification of Aspergillus ochraceus and Aspergillus westerdijkiae in grapes and green coffee beans by real-time quantitative PCR. , 2009, International journal of food microbiology.

[8]  M. Hamdy,et al.  EFFECT OF STAPHYLOCOCCUS INFECTION ON ACID AND ALKALINE PHOSPHATASE IN BRUISED POULTRY TISSUE , 1970 .

[9]  C. Vázquez,et al.  Specific detection of Aspergillus carbonarius by SYBR Green and TaqMan quantitative PCR assays based on the multicopy ITS2 region of the rRNA gene. , 2009, FEMS microbiology letters.

[10]  P. Elizaquível,et al.  Comparison of four commercial DNA extraction kits for PCR detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in fresh, minimally processed vegetables. , 2008, Journal of food protection.

[11]  N. H. Spliid,et al.  Real-time PCR for quantification of eleven individual Fusarium species in cereals. , 2009, Journal of microbiological methods.

[12]  N. Paster,et al.  Detection of aflatoxigenic molds in grains by PCR , 1996, Applied and environmental microbiology.

[13]  J. Frisvad,et al.  A reappraisal of fungi producing aflatoxins , 2009 .

[14]  R. Aznar,et al.  Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes. , 2008, International journal of food microbiology.

[15]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[16]  A. Ciancio,et al.  Detection and quantification of Aspergillus section Flavi spp. in stored peanuts by real-time PCR of nor-1 gene, and effects of storage conditions on aflatoxin production. , 2010, International journal of food microbiology.

[17]  K. Ehrlich,et al.  What does genetic diversity of Aspergillus flavus tell us about Aspergillus oryzae? , 2010, International journal of food microbiology.

[18]  J. J. Córdoba,et al.  Development of real-time PCR methods to quantify patulin-producing molds in food products. , 2011, Food microbiology.

[19]  A. Dobson,et al.  The use of reverse transcription-polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439. , 2000, International journal of food microbiology.

[20]  M. Rodríguez,et al.  Composition and toxigenic potential of the mould population on dry-cured Iberian ham. , 1996, International journal of food microbiology.

[21]  J. J. Córdoba,et al.  Production of cyclopiazonic acid by Penicillium commune isolated from dry-cured ham on a meat extract-based substrate. , 2002, Journal of food protection.

[22]  N. Magan,et al.  Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature. , 2009, International journal of food microbiology.

[23]  John E. Linz,et al.  Clustered Pathway Genes in Aflatoxin Biosynthesis , 2004, Applied and Environmental Microbiology.

[24]  J. Cary,et al.  Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi. , 2005, Mycologia.

[25]  I. Skaar,et al.  Mould contamination in production of semi-hard cheese. , 2004, International journal of food microbiology.

[26]  Björn Sjögreen,et al.  The real-time polymerase chain reaction. , 2006, Molecular aspects of medicine.

[27]  C. Juan,et al.  Limited survey for the occurrence of aflatoxins in cereals and poultry feeds from Rabat, Morocco. , 2007, International journal of food microbiology.

[28]  J. Frisvad,et al.  Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. , 2008, International journal of systematic and evolutionary microbiology.

[29]  W. Abu Al‐Soud,et al.  Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples , 1998, Applied and Environmental Microbiology.

[30]  C. Woloshuk,et al.  Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium and Fusarium , 2009 .

[31]  N. L. Glass,et al.  Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes , 1995, Applied and environmental microbiology.

[32]  E. Topp,et al.  A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure. , 2003, Journal of microbiological methods.

[33]  W. Holzapfel,et al.  Detection of aflatoxinogenic fungi in figs by a PCR reaction. , 1997, International journal of food microbiology.

[34]  N. H. Spliid,et al.  Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels. , 2008, Journal of microbiological methods.

[35]  Alicia Rodríguez,et al.  Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods. , 2011, International journal of food microbiology.

[36]  John E. Linz,et al.  Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus , 2004, Archives of Microbiology.

[37]  Kirk M. Ririe,et al.  Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. , 1997, Analytical biochemistry.

[38]  R. Geisen,et al.  Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods. , 2003, International journal of food microbiology.

[39]  C. Vázquez,et al.  Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR. , 2011, International journal of food microbiology.

[40]  A. Logrieco,et al.  Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes. , 2006, International journal of food microbiology.

[41]  A. Chandrashekar,et al.  Detection of aflatoxigenic fungi in selected food commodities by PCR , 2005 .

[42]  L. Giannuzzi,et al.  Modelling of aflatoxin production by Aspergillus parasiticus in a solid medium at different temperatures, pH and propionic acid concentrations. , 2002 .

[43]  A. Erdoğan.  The aflatoxin contamination of some pepper types sold in Turkey. , 2004, Chemosphere.

[44]  H. Hajjaj,et al.  Aflatoxin genes and the aflatoxigenic potential of Koji moulds , 2001, Applied Microbiology and Biotechnology.

[45]  N. Heutte,et al.  Evaluation of fungal contamination and mycotoxin production in maize silage , 2009 .

[46]  F. Rojas,et al.  Mycoflora and toxigenic Aspergillus flavus in Spanish dry-cured ham. , 1991, International journal of food microbiology.