A Unique Rapid Detection and Quantification Assay for Total Count of Yeasts and Molds in Dairy Products Based on Multiplex Real-time PCR

Introduction: Ubiquitous in the environment, yeasts and molds can easily contaminate foods and become the predominant spoilers, particularly in dairy products. Thus, their presence and amount are regularly monitored. Conventional methods can take ≤14 days, and current rapid methods require multiple days for an answer. A same-day test would significantly benefit the dairy industry economically with faster product release.

Purpose: Validation of the sensitivity, robustness and specificity of a lyophilized real-time PCR assay (the foodproof Yeast & Mold Quantification LyoKit) for detection and quantification of yeasts and molds in dairy products. 

Methods:  Ten different dairy sample types were diluted 1:10 and spiked with yeasts and molds at specific concentrations of ≤6 x 10³ cfu/g. Homogenized sample (800 µl) was treated with Reagent D for live/dead cell differentiation.  Only live cell DNA was extracted and real-time PCR performed. Live/dead differentiation efficiency, sensitivity (DNA and cell spiked samples), specificity (inclusivity and exclusivity), and robustness were determined for the new foodproof Yeast and Mold Quantification LyoKit, and compared to the classical ISO-method (ISO 6611).

Results:  Genomic DNA from 15 yeast and mold species was tested with 11 replicates. 100% of replicates were positive, even at 0.39 GE in all dairy samples. All sample types spiked with 10² and 10³ cfu/g Yarrowia lipolytica, Aspergillus niger, Candida kefyr, and Hypopichia burtonii showed appropriate positive signal. All samples spiked with 6000, 600, 60 and 6 cfu/g showed positive results with a deviation of <1 ct. Thus, quantification was successful (LOD of < 10 cfu/g). Comparison to ISO 6611 showed good correlation independent of matrix type. Specificity results showed 100% success for inclusivity (290 strains from 260 species) and exclusivity testing (>60 bacteria, plants and mammal cells).

Significance:  Validation of the new commercial kit showed results in >4 hours equivalent to or better than ISO 6611 for the detection and quantification of yeasts and molds in dairy products. This kit has proven to be the first rapid same day method available to accurately quantify yeast and molds, particularly in dairy samples.