Development and evaluation of a real-time quantitative PCR assay forAspergillus flavus

• DOI: 10.3852/08-022
• Publication Date: 2008-10-27T16:32:37Z
• Authors: Patricia Cruz; Mark P. Buttner
• Keywords: Primer (cosmetics); TaqMan; Serial dilution; genomic DNA

Aspergillus flavus is a ubiquitous mold and the most common contaminating foodstuffs. Many strains of A. produce aflatoxins. In addition it an allergen opportunistic pathogen animals plants. often underestimated in traditional culture analyses due to expertise required cost associated with speciating members genus Aspergillus. The goal this study was develop validate primer probe set for rapid detection quantitation pure using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located genome target organism by sequence comparison GenBank database, several candidate oligonucleotides identified from scientific literature potential use TaqMan® QPCR technology. Three sets designed validated specificity sensitivity laboratory experiments. Initial screening test performed seven isolates selected nontarget fungi. Specificity testing conducted set, which amplified all nine tested, including aflatoxin producing strain. primers did not amplify extracted 39 other fungal species (comprising 16 genera), 18 six Penicillium species. No amplification human or bacterial observed; however cross-reactivity observed oryzae. PCR analysis dilutions internal positive control demonstrated that 67% samples assayed contained inhibitors. assay capable results less than 1 h after extraction. research demonstrate capabilities enhanced enumeration fungi significance health.