Technologies for Quantifying Contamination of Foodstuffs with Aflatoxin-Producing Aspergillus

Non-Technical Summary:<br/>
To control contamination of stored grain and other foodstuffs with toxin-producing molds, a reliable, sensitive, and inexpensive means is needed for assessing the extent of contamination of grain or food before transfer to storage and for monitoring development or increase in mold contamination during storage. The most economically important group of storage molds is within the genus Aspergillus, which is also the source of aflatoxin B1, the most potent naturally occurring carcinogen known. We have developed a highly efficient means of extracting Aspergillus spores from stored commodities, and this research proposes to develop a technique for quantitative assessment of Aspergillus contamination, using both simple, easily applied microbiological assays and more definitive molecular assays that depend upon specific, differentiating DNA sequence information.
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Approach:<br/>
(Objective 1) An objective of this proposed research is to develop a grain or foodstuff extraction and analysis kit that consists of prepackaged extraction reagent, a simple and inexpensive concentration apparatus (such as a hand-operated plastic syringe with an in-line filter disc), plastic bulb pipettes, and a Petrifilm-based quantitative assay film that contains custom-prepared selective growth medium and indicator dyes to enhance visualization of mold growth on the film. The assays will be made sensitive over a range of mold contamination with serial dilutions of the extracted samples, and they will be made quantitative by counting mold colonies on surfaces of the Petrifilm cultures. The Petrifilm technology may be easily altered for use of growth media that are devised for specific organisms, such as yeasts and food-storage molds. Using field-contaminated peanuts and maize kernels (along with laboratory-generated samples of known contamination), we will determine what levels of Aspergillus contamination represent safe levels, given anticipated storage conditions (moisture, temperature, and time) and what levels of contamination represent a hazard.
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(Objective 2) The goal of the second part of this project is to devise a sensitive and highly species-specific assay for research into storage mold contamination, concentrating on species of Aspergillus that are known to produce aflatoxins in foodstuffs. We will apply known quantities of storage mold spores to grain samples, extract these spores with the paraffinic hydrocarbon Soltrol, and determine the range of sensitivity of these molecular assays. Whereas the goal of this project is to detect and quantify A. flavus contamination in foodstuffs, another species, A. oryzae, will be used (a) in laboratory experimentation with artificially contaminated grain and (b) as an internal control for efficiency and specificity of extraction of A. flavus from grain samples naturally contaminated with A. flavus. The basis of the molecular assays will be the polymerase chain reaction, or PCR, which allows quantitative detection of extremely small numbers of specific cells. The DNA will be extracted from storage mold spores . It is this a 257-bp deletion in the a?T gene that provides a suitable site for differentiation via PCR between A. ?avus and A. oryzae. DNA oligonucleotide primers (both forward and reverse) have been designed for this gene and the deletion, as well as all other sequence differences between the two species and for beta-tubulin and the universal fungal chitin synthetase gene (Tominaga et al., 2006). We will use PCR to determine whether a specific DNA sequence is present in a sample and the number of its copies in the sample. The PCR quantification will depend upon measurement of either an absolute number of copies or a relative amount when normalized to a known amount of DNA in spores present in a control grain sample extraction. The target DNA is quantified by measuring increase in DNA concentration during the exponential amplification phase against the number of PCR cycles.