Quantification of Yeasts and Molds in Dairy Products By Real-Time PCR

Introduction:   Since yeasts and molds are ubiquitous in the environment, foods can be easily contaminated through raw material, inadequately sanitized equipment, or simply by airborne contaminants. When conditions for bacterial growth are less favorable, yeasts and molds may become predominant spoilers in dairy products. Conventional methods for the detection of yeasts and molds take up to 14 days. During this period, the product is not available for the market. A rapid test would prolong the distribution time.

Purpose: To develop the foodproof^® Yeast & Mold Quantification LyoKit; a fast and sensitive assay for the detection and quantification of yeasts and molds in dairy products, with a time to result of less than 5 hours.

Methods:  Food samples were diluted 1:10 in a stomacher bag. After homogenization, 800 µl of sample was used for DNA extraction, which included live/dead differentiation and a mechanical disruption step. Purified DNA was used directly for qPCR. The efficiency of live/dead differentiation, sensitivity, specificity, and robustness were determined. The assay was compared to the ISO-method.

Results:  Genomic DNA from 15 different species was tested in 11 replicates. Even at the lowest concentrations (0.39 GE/reaction), 100% of all replicates were positive. Ten different samples were spiked with 10² and 10³ cfu Yarrowia lipolytica/g . Deviation of the threshold cycle was less than one Ct. Samples spiked with 6000, 600, 60, and 6 cfu/g showed positive results. The lower detection limit of < 10 cfu/g was fulfilled. Comparison of quantification between ISO-method and PCR of spiked samples showed good correlations. Specificity (inclusivity and exclusivity) is 100%. Two hundred sixty (260) species were tested, positively, with the kit. More than 60 bacteria, plants, and mammal cells were tested negative.

Significance: The foodproof^® Yeast and Mold Quantification  LyoKit is the first qPCR test on the market, which detects and quantifies yeasts and molds, in a single test, in less than 5 hours.