Date of this Version
Nguyen, B. (2017). Development of a Rapid Detection and Quantification Method for Yeasts and Molds in Dairy Products (Masters Thesis). University of Nebraska-Lincoln. Food Science and Technology. 99pp.
A rapid quantitative PCR (qPCR) method was developed for the detection and quantification of fungi that are potentially present in dairy commodities. Genes of interest that were considered and used in the method development were the following: 18S rRNA, actin, beta-tubulin, and elongation factor 1-alpha. The following organisms were screened in this method development: Galactomyces candidus, Debaryomyces hansenii, Yarrowia lipolytica, Penicillium roqueforti, Penicillium verrocosum and Cladosporium cladosporioides. The developed method has a standard curve based on the organism, Galactomyces candidus, and the primers based on the elongation factor 1-alpha gene. Using this yeast and primers, this method can detect fungal counts above 102 CFU/mL. This method was compared to traditional plate counting on DRBC agar and currently available commercial methods (3M Rapid Yeast and Mold Petrifilms, Hygiena Qualicon BAX PCR Assay for Yeasts and Molds, and BIOTECON’s FoodProof Yeast and Mold Quantification LyoKit -5’Nuclease- RP). The method comparison was performed at 4 set concentrations (106, 104, 102, and 0) in culture material and inoculated dairy samples. After this method comparison was performed in triplicate by two technicians, a survey testing 38 assorted dairy products (fluid milk, cottage cheese, yogurt, sour cream, and cheese) was performed. 16 of the 38 samples exhibited amplification using the developed PCR method and with an estimated fungal presence in these samples between 102 and 105 CFU/mL. Of all the methods tested in the survey and the comparison work, the 3M Petrifilm provided the most consistent results that were comparable to the standard plate counting method on DRBC. The developed qPCR method was comparable in performance to the commercially available BIOTECON kit.
Advisor: Andréia Bianchini