An uncertainty evaluation method for the microbiological determination of biotin in infant formula was developed in order to control the quality of test results and improve the detection accuracy, a method for uncertainty evaluation of milk powder by method was established. According to the requirements of the Evaluation and Expression of Uncertainty in Measurement (JJF 1059.1—2012) and the Guide to the Evaluation and Expression of Uncertainty in Measurement (GB/T 27418—2017), the uncertainty components were estimated for the determination of biotin in infant formula by the method specified in the National Food Safety Standard Determination of Biotin in Foods (GB 5009.259—2016). It was found that the greatest uncertainty resulted from standard curve fitting and repeatability test. The uncertainty for the biotin content of three infant formula samples was as follows: sample A (19.87 ± 2.84) μg/100 g, B (30.35 ± 4.41) μg/100 g, and C (53.15 ± 7.15) μg/100 g (in the 95% confidence interval, coverage factor (k) = 2). As the content of biotin in samples increased, the expanded uncertainty also increased.

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.

A rapid detection method for Salmonella in fermented milk was developed by helicase-dependent isothermal DNA amplification (HDA). In this method, the invA gene sequence of Salmonella was used as the target gene to design specific primers, and the concentrations of UvrD helicase and T4 gp32 in the reaction system were optimized to establish the optimal reaction system. The HDA method was used to directly detect Salmonella in fermented milk, and the amplification products were detected by electrophoresis to verify the specificity of this method. The results showed that the proposed method exhibited high specificity for Salmonella in fermented milk. The optimized reaction system contained 0.10 μg of UvrD helicase and 5.0 μg of T4 gp32 per 50 μL of sample. The amplification product was consistent with the designed sequence length (304 bp). The detection limit was 2.6 × 102 CFU/g. The method can meet the requirements for the rapid detection of Salmonella in fermented milk, and thanks to its high sensitivity and easy operation, it can be used as a basic method for rapid detection of Salmonella in fermented milk.

A helicase-dependent isothermal DNA amplification (HDA) assay was developed for the rapid and accurate detection of Gluconacetobacter in yogurt.A pair of specific oligonucleotide primers according to the 16S ribosomal RNA gene (GenBank accession number:HQ677466.1) of Gluconacetobacter was designed,and the optimal concentration of UvrD helicase and T4 gp32 in the reaction system were determined.The HAD method enabled direct detection of Gluconacetobacter in yogurt.The specificity of the method was tested by amplification and electrophoresis of various strains in yoghurt using the HAD system.The amplification product by HDA was the same as the designed gene fragment (101 bp).The sensitivity of HDA was 102 CFU/g.The optimal reaction system was established by the use of 0.1 μg of UvrD helicase and 5.0 μg of T4 gp32.In conclusion,the method was sensitive and time saving.

Objective: A loop-mediated isothermal amplification (LAMP) assay was designed for rapid and accurate detection of Bacillus cereus in yogurt. Methods: Two pairs of oligonucleotide primers exclusively amplifying the hblA gene of Bacillus cereus were designed and Bacillus cereus could be directly detected by LAMP in yogurt, and the amplification products were detected by electrophoresis. Results: Detection of Bacillus cereus in yogurt by the LAMP assay was highly specific, the sensitivity was 6.4 CFU/mL, and the detection limit was 21 CFU/mL. Conclusion: The LAMP was fast, sensitive and useful for the detection of Bacillus cereus in yogurt.