Development and Application of PMA-qPCR Method for Detecting Viable Yeasts in Dairy Products

This experiment was aimed to accurately determine the total number of viable yeast strains in dairy. The best working conditions of PMA for heat-damaged yeast in dairy products was investigated to removal of heat-damaged DNA. Primers Y3 and Y4 were designed for the 26S rDNA D1/D2 region sequences of three yeasts isolated from dairy and real-time quantitative PCR was used to perform sequence amplification. The copy number and Ct value of recombinant plasmid pMD-18T-26S were calculated,then the standard curve was established on the coordinate axis,and the PMA-qPCR detection method was established. The amount of live yeast in the actual sample was enriched and quantitatively detected. The PMA-qPCR method could meet the standards of the yeast maximum limit value of 1×10² CFU/mL in foods specified by the national standard with good repeatability,corresponding to a Ct value of 36.67±0.43,and the total time taken was 6~7 h. The amount of yeast could be determined. Accurate and rapid quantitative analysis could provide valuable reference for the identification and monitoring of spoilage microorganisms in dairy products.