"Currently, there are few commercial rapid methods to detect molds and their spores in agricultural commodities, grains, and foods."

Immunocapture real-time PCR to detect mycotoxigenic mold spores in grains

Investigators: Maribeth Cousin (Department of Food Science), Charles Woloshuk (Department of Botany and Plant Pathology)

Project Report 2006 - 2007

» Download Project Report 2006 - 2007

Project Rationale

Currently, there are few commercial rapid methods to detect molds and their spores in agricultural commodities, grains, and foods. In previous research, a protocol was developed to identify Fusarium species that produce two major mycotoxins, including fumonisins and trichothecenes. The antibody-based method was developed for Fusarium species to capture antigens of these mycotoxin-producers, which was then combined with a real-time PCR assay that was based on species-specific and genus-specific primers to identify the Fusarium species. The previous research was limited in spore capture efficiency because the Fusarium spores were difficult to lyse for DNA release. We have designed this new project to help resolve these limitations by: 1) studying physical, enzymatic, and mechanical methods to break mold spores to release DNA for use in real-time PCR; and 2) incorporating the method into a immunocapture-qPCR method that uses antibodies produced against Fusarium graminearum and F. verticillioides and primers that are specific for the Tri6 gene involved in trichothecene biosynthesis and for the Fum1 gene involved in fumonisin biosynthesis. This project was also designed to develop a library of PCR primers to other mycotoxigenic genera (Aspergillus that produce aflatoxins and ochratoxin and Penicillium that produce ochratoxin and patulin) for real-time PCR and to use these primers multiplex PCR formats to detect all major mycotoxin producers in the same assay. Antibodies to aflatoxin-producing molds (Yong and Cousin, 2001) and Penicillium species (Tsai and Cousin, 1990) were produced in earlier CFSE-funded research.

Project Objectives

  • Develop primer sets to detect Aspergillus and Penicillium species.
  • Determine specificity and sensitivity of primer sets and multiplex format.
  • Experiment with different methods to break mold spores of Fusarium species.
  • Optimize the capture of mold spores and release of DNA and use to detect Fusarium species in foods and grains.

Project Highlights

We designed PCR probes specific to species of Aspergillus (three probes), Fusarium (one probe), and Penicillium (two probes). Multiple genus-probes are required to capture minor groups or individual species that have slight differences in their DNA sequence. We have determined the optimum PCR conditions to achieve specificity under multiplex conditions and developed quantitative curves. The linear range of the probes was between 0.01 ng and 1000 ng of target DNA. Thus far, the major probes for each genus have been tested.

Annual Reports

Investigators

  • Maribeth Cousin
  • Charles Woloshuk