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Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the non-ribosomal peptide synthetase (otanpsPN) gene involved in OTA biosynthesis. Seventy five mold strains representing OTA producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1x10(4) to 10conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between 1 and 10conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g(-1) per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A multiplex polymerase chain reaction (PCR) procedure based on fliC(h7) and rfbE genes was developed for the detection of Escherichia coli O157:H7 in raw pork meat and ready-to-eat (RTE) meat products. Two different DNA extraction procedures were evaluated for application on meat products. MasterPure™ DNA Purification kit in combination with immunomagnetic separation was found to be the best method in a meat system. The optimized PCR included an enrichment step in brilliant green bile 2% broth at 37 °C. This method was applied to artificially inoculated meat and RTE meat products with different concentrations of E. coli O157:H7. The results indicate that the PCR assay developed could sensitively and specifically detect E. coli O157:H7 in raw pork meat and RTE meat products in approximately 10h, including a 6h enrichment step. Thus, this method could be proposed for screening E. coli O157:H7 in raw pork and RTE meat products., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Abstract: Accumulation of ochratoxin A (OTA) on the surface and to a 0.5cm depth of dry-cured Iberian ham after initial fungal growth was investigated. For this, 20 dry-cured Iberian hams from the drying stage showing incipient fungal growth on the surface were analyzed. In addition, the presence of OTA-producing molds was examined on the surface of the hams by real-time quantitative PCR (qPCR) based on the otanpsPN gene. Quantification of specific OTA-producing molds, such as Penicillium nordicum and Penicillium verrucosum was also achieved on the hams by specific qPCR methods. Ten of 20 dry-cured hams showed OTA at higher levels than those established by legal regulation. OTA was even detected in the deep section of hams. OTA-producing molds ranged from 1.5 to 7.3logcfu/cm2. Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum. [Copyright &y& Elsevier]

Abstract: The growth of aflatoxin B1 (AFB1), ochratoxin A (OTA) and patulin producing strains and the hazard of their mycotoxins production in dry-cured ham were evaluated. In addition, effectiveness of real-time quantitative PCR (qPCR) for the detection and quantification of toxigenic moulds in this product before mycotoxins production was analyzed. For this, slices of dry-cured ham were inoculated in separate assays with AFB1, OTA and patulin producers and incubated at 97% and 84% relative humidity (RH) for 21 days at 25 °C. Higher counts were reached by AFB1 and patulin producing species at 97% RH, and by OTA producing species at 84% RH. The production of AFB1, OTA and patulin on dry-cured ham slices was demonstrated. The estimation of toxigenic moulds by qPCR was highly correlated with the counts obtained by plating in culture media. All the qPCR protocols assayed detected and quantified toxigenic moulds in the hams before mycotoxins were produced. Thus, qPCR should be considered for a reliable and rapid quantification of toxigenic moulds in dry-cured ham. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Quantification 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 β-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 Chelex-100 resin and final extraction with the EZNA kit was selected, since the extracted DNA showed good amplification by conventional PCR for β-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. [Copyright &y& Elsevier]

Abstract: A multiplex real-time PCR (qPCR) method to quantify aflatoxin, ochratoxin A (OTA) and patulin producing molds in foods was developed. For this, the primer pairs F/R-omt, F/R-npstr and F/R-idhtrb and the TaqMan probes, OMTprobe, NPSprobe and IDHprobe targeting the omt-1, otanpsPN and idh genes involved in aflatoxin, OTA and patulin biosynthesis, respectively, were used. The functionality of the developed qPCR method was demonstrated by the high linear relationship of the standard curves constructed with the omt-1, otanpsPN and idh gene copies and threshold cycle (Ct) values for the respective producing molds tested to quantify aflatoxin, OTA and patulin producing molds. The ability of the optimized qPCR protocol to quantify producing molds was evaluated in different artificially inoculated foods (fruits, nuts, cereals and dry-ripened meat and cheese products). Efficiency values ranged from 81 to 110% in all inoculated foods. The detection limit was between 3 and 1logcfu/g for aflatoxin, OTA and patulin producing molds. The developed multiplex qPCR was shown be an appropriate tool for sensitive quantification of growth of toxigenic fungi in foods throughout the incubation time. Thus, the multiplex qPCR is a useful, rapid and efficient method to quantify simultaneously aflatoxin, OTA and patulin producing molds in food products. [Copyright &y& Elsevier]

Abstract: The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)4 and (GAC)5 was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening. [Copyright &y& Elsevier]

A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/µl in 150 µl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and nontoxigenic molds, a procedure of great interest in food safety. [ABSTRACT FROM AUTHOR]

Abstract: In this work the implantation of a protective culture of Penicillium nalgiovense on commercial dry-fermented sausages “salchichón” and its effect over presence of mycotoxin-producing moulds belonging to contamination origin was evaluated. In addition, the suitability of real-time quantitative PCR (qPCR) as a rapid and sensitive method to test implantation of protective culture throughout the “salchichón” processing was also tested. Dry-fermented sausages “salchichón” inoculated with a non-toxigenic protective P. nalgiovense and subjected to three different commercial ripening processes were analysed. At first, ability of P. nalgiovense strain to avoid growth of an ocratoxin A (OTA)-producing strain and its mycotoxin production in a controlled model system was demonstrated. P. nalgiovense was quantified by a qPCR designed on the basis of the ITS region and values higher than 106 ufc/cm2 in both inoculated and non-inoculated “salchichón” were obtained. This technique should be considered a good tool to verify the implantation of protective culture of P. nalgiovense. Producing moulds of aflatoxins, OTA, patulin, sterigmatocystin and verrucosidin and the corresponding mycotoxins were not detected in any dry-fermented sausages tested, including those non-inoculated ones. Thus, presence of P. nalgiovense is inhibiting growth of toxigenic moulds. Utilization of a non-toxigenic fungal protective culture in dry-fermented sausage “salchichón” processing should be considered as a good tool in the preventive programmes to avoid growth of toxigenic moulds. [Copyright &y& Elsevier]

Abstract: A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1–4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain. [Copyright &y& Elsevier]

Abstract: Rapid and specific detection of viable Escherichia coli O157:H7 cells in ready-to-eat (RTE) meat products, by duplex quantitative PCR (qPCR) procedures with mRNA and SYBR Green and TaqMan methodologies were developed. Specific primers and probes were designed based on the serotype of E. coli O157:H7, fliCh7 and rfbE genes. No cross-reactivity with other microorganisms was observed. The detection limit of the assays was 101 or 102 CFU/g for artificially contaminated meat products, and after a 4h enrichment period at 37°C, the detection limit decreased to about 1CFU/g. Time-to completion of the assay was approximately 8h. Thus, these qPCR methods offer a useful, rapid and efficient tool for screening viable E. coli O157:H7 in RTE meat products. This tool could also be proposed for monitoring these foodborne pathogens in HACCP programs. [Copyright &y& Elsevier]

Abstract: Verrucosidin, which is a tremorgenic mycotoxin responsible for neurological diseases, has been detected in different dry-ripened foods as consequence of the growth of toxigenic molds. To improve food safety, the presence of verrucosidin producing molds in these kind foods should be quantified. The aim of this study was to design a duplex real-time PCR (qPCR) protocol based on TaqMan methodology with an internal amplification control (IAC). Eleven verrucosidin producing and 11 non producing strains belonging to different species often reported in food products were used. Verrucosidin production was tested by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography–mass spectrometry (HPLC–MS). A primer pair (VerF1/VerR1) and a TaqMan probe (Verprobe) were designed from the SVr1 probe sequence of a verrucosidin producing Penicillium polonicum. The conserved regions of the β-tubulin gene were used to design primers (TubF1/TubR1) and probe (Tubprobe) of the non-competitive IAC. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves which relating Ct values and DNA template of the tested verrucosidin producers using the verrucosidin and IAC primers. The ability to quantify verrucosidin producers of the developed TaqMan assay in all artificially inoculated food samples was successful, with a minimum detection limit of 1 log cfu per gram of food. This qPCR protocol including an IAC could be very useful to quantify verrucosidin producing molds in dry-ripened foods avoiding false negative results. This method should be proposed to monitor the target molds in HACCP programs to prevent the risk of verrucosidin formation and consequently avoid its presence in the food chain. [Copyright &y& Elsevier]

Abstract: Patulin is a secondary toxic metabolite with important health effects. Several mould species of Penicillium and Aspergillus genera associated with patulin production have been detected in food products. Thus, specific and sensitive methods to detect patulin producing moulds are needed. The aim of this work was to develop a polymerase chain reaction (PCR) method to detect patulin producing moulds in food. 34 patulin producing and 30 non-producing strains belonging to the main species usually reported in food products were used. Patulin production was firstly evaluated by mycellar electrokinetic capillary electrophoresis and high-pressure liquid chromatography-mass spectrometry in all tested strains. Biosynthesis was also used to develop PCR primers derived from the genes involved in patulin. By means of a primer pair based on the isoepoxydon dehydrogenase (idh) gene, a 496-bp amplicon was specifically detected in all the mould strains previously confirmed as patulin producing, regardless of their genus and species. With the developed method it was possible to detect down to 0.5 ng of pure DNA from producing strains and from 1.8 × 102 to 2.7 × 103 conidia g−1 in artificially inoculated foods. No relevant PCR inhibition due to food matrices was observed. The PCR protocol developed could be considered as an appropriate tool to detect patulin producing moulds in food products. [Copyright &y& Elsevier]