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.

Development of a PCR protocol to detect aflatoxigenic molds in food products.

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal ß-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.

Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods.

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.

Development of real-time PCR methods to quantify patulin-producing molds in food products.

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.

Development of PCR assays for detection of Escherichia coli O157:H7 in meat products.

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.