Annual Report - Center for Food Safety Engineering - Purdue ...

Cousin and WoloshukImmunocapture real-time PCR to detectmycotoxigenic mold spores in grainsInvestigators: Maribeth A. Cousin (Department of Food Science), Charles P. Woloshuk (Department of Botany and Plant Pathology)Project RationaleCurrently, there are few commercial rapid methods to detectmolds and their spores in agricultural commodities, grains,and foods. In previous research, a protocol was developed toidentify Fusarium species that produce two major mycotoxins:fumonisins and trichothecenes. This antibody-based method wasdeveloped for Fusarium species to capture antigens of thesemycotoxin-producers, which were then combined with a realtimePCR assay that was based on species-specifi c and genusspecific primers to identify the Fusarium species. The effi ciencyof spore capture was limited in the previous research becausethe Fusarium spores were diffi cult to lyse for DNA release. Weproposed this new research to help resolve that limitation by:(1) studying physical, enzymatic, and mechanical methods tobreak mold spores to release DNA for use in real-time PCR, and(2) incorporating the method developed in objective 1 into theimmunocapture-qPCR method that uses antibodies producedagainst F. graminearum and F. verticillioides and primers that arespecifi c for the Tri6 gene involved in trichothecene biosynthesisand for the Fum1 gene involved in fumonisin biosynthesis. Inaddition, we proposed to develop a library of PCR primers toother mycotoxigenic genera (Aspergillus that produce afl atoxinsand ochratoxin and Penicillium that produce ochratoxin andpatulin) for real-time PCR, and to use these primers in multiplexPCR formats to detect all major mycotoxin producers in thesame assay. Antibodies to afl atoxin-producing molds andPenicillium species were produced in earlier research.• Determine the specifi city and sensitivity ofprimer sets and multiplex format.• Optimize the capture of mold spores and release of DNAused to detect Fusarium species in foods and grains.Project HighlightsA procedure was developed and optimized using lyticase(Sigma: 5263) to extract DNA from conidia of Fusariumgraminearum and Fusarium verticillioides for use in real-timequantitative PCR (qPCR). This was important because themethods used to extract DNA from other microorganisms do notwork for fi lamentous fungi. Lyticase was mixed with buffer andmercaptoethanol, incubated at 37°C for four or six hours for F.graminearum and F. verticillioides, respectively, and shaken ina bead-beater to physically disrupt the conidia before analyzingwith real-time qPCR. By this method, a minimum of 10 conidiaof F. graminearum and 1000 conidia of F. verticillioides could bedetected.Project Objectives• Develop primer sets to detect Aspergillusand Penicillium species.• Experiment with different methods to breakmold spores of Fusarium species.6Center for Food Safety Engineering“This antibody-based method was developed for Fusarium species to capture antigens ofthese mycotoxin-producers, which were then combined with a real-time PCR assay...”