title: Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.
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.
authors.affiliation: Higiene y Seguridad Alimentaria, Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n., 10071 Cáceres, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Rodríguez
authors.forename: Mar
authors.initials: M
authors.lastname: Rodríguez
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Luque
authors.forename: Alberto
authors.initials: A
authors.lastname: Martín
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Córdoba
mesh_terms: D000348:Aflatoxins / Q000032:analysis*; D010367:Arachis / Q000382:microbiology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017931:DNA Primers; D004271:DNA, Fungal / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005658:Fungi / Q000254:growth & development / Q000302:isolation & purification*; D008461:Meat Products / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017365:Spices / Q000382:microbiology
chemical_list: D000348:Aflatoxins; D017931:DNA Primers; D004271:DNA, Fungal