In order to improve the detection efficiency of molds and yeasts in low-temperature yogurt, primers and probes were designed according to the sequences of the conserved gene elongation factor-1α (EF-1α) for molds and yeasts, respectively. A real-time fluorescence quantitative polymerase chain reaction (real-time PCR) method with TaqMan probe was established for the detection of molds and yeasts, respectively. The genomic DNA of fungi and lactic acid bacteria commonly found in low-temperature yogurt, were used as amplification templates for specificity testing. The sensitivity and repeatability were also tested, and the standard curves were constructed. The results showed that the designed primers and probes had good specificity; the sensitivity of the method was 101 and 102 CFU/mL for yeasts and molds, respectively; the coefficients of variation within and between groups in the repeatability test were less than 2%, indicating good reliability and stability; the correlation coefficients of the standard curves were higher than 0.99. Therefore, the real-time PCR method enables the rapid detection of molds and yeasts in low-temperature yogurt.

A droplet digital polymerase chain reaction (ddPCR) method for the quantitative detection Acetobacter aceti in fermented milk was established. Specific primers and probes were designed according to the internally transcribed spacer (ITS) gene sequence of Acetobacter aceti, and annealing temperature was optimized. The specificity of the method was verified by applying it on various strains, the limit of detection (LOD) was determined for artificially inoculated Acetobacter aceti, and the absolute quantification was systematically investigated by comparing the results of ddPCR and the counting results. The experimental results showed that the optimal annealing temperature was 54.6 ℃, the method had strong specificity and high sensitivity, and the LOD was 7.2 × 101 CFU/mL. The quantitative deviation rate was 23.73%. This method can meet the demand for quantitative detection of Acetobacter aceti in fermented milk.