This study aimed to establish a rapid method for identifying three common foodborne pathogenic Gram-negative bacteria (Vibrio parahaemolyticus, Enterobacter cloacae, and Acinetobacter baumannii). We explored the effects of different pretreatment methods, media, and culture durations on the results of bacterial identification using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We optimized the MALDI-TOF/TOF MS (tandem TOF MS) method with respect to culture medium and time. Formic acid-acetonitrile extraction was found to be the most suitable pretreatment method. The single-stage TOF MS spectra showed significant differences in identification scores, the number of characteristic peaks, and morphology among strains grown on different media. The number of major ion peaks decreased with culture time, leading to reduced matching accuracy. The tandem TOF MS spectra showed no significant changes in the characteristic peaks of strains at different culture times, indicating that the tandem TOF MS method was less affected by the external environment, exhibiting higher specificity. The single-stage TOF MS method had high accuracy for identifying strains cultured in specific environments for short durations. However, as the culture environment changed and the culture time increased, the results of single-stage TOF MS became unstable, while those of tandem TOF MS changed little. When they were applied to identify Gram-negative bacteria, compared with single-stage TOF MS, tandem TOF MS gave more stable results with fewer interfering peaks, thereby serving as an effective supplement to the existing microbial identification methods.

The rapid identification of Salmonella, a significant foodborne pathogen, is of significant importance. To establish a new method for identifying Salmonella based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), this study optimized culture medium type, incubation time, bacterial lysis methods, and instrumental parameters. The results showed that compared to the national standard method GB/T 33682-2025 General microorganism identification method with matrix-assisted laser desorption/ionization time of flight mass spectrometry, the recovery rate increased from 54.7% to 84.7% after replacing the culture medium with nutrient agar. Increasing the incubation time from 24 to 48 h increased the average identification score by 7.0%, from 2.203 to 2.355. Optimizing lysis methods elevated the average identification score by 19.0%, from 1.898 to 2.259. When the instrumental parameters were adjusted to 50% laser energy and a laser wavelength of 370 nm, the average identification score increased to 2.211. Compared to its original version specified in GB/T 33682-2025, the established MALDI-TOF/TOF MS method exhibited improved accuracy and reliability in Salmonella identification.

A rapid detection method based on helicase-dependent isothermal DNA amplification (HDA) was established for Lactobacillus plantarum in fermented milk. Specific primers were designed according to the scrB gene sequence of Lactobacillus plantarum (Genbank Accession NO. AJ579541.1), and the optimal concentrations of UvrD helicase and T4 gp32 in the reaction system were determined through experiments. The limit of detection (LOD), specificity, consistency and stability of the proposed method were evaluated by use of L. plantarum-spiked samples, amplification of various strains, and electrophoresis of amplified products and sequence alignment analysis, respectively. The results showed that the optimized of UvrD helicase and T4 gp32 in the reaction system were found to be 0.15 and 5.0 μg, respectively. The HDA method had high specificity with no amplification of other strains tested. The detection limit was 2.8 × 10¹ CFU/g. The amplified product was consistent with the designed sequence length (273 bp) and the sequence homology was 100%. In conclusion: this method is rapid, simple, sensitive and suitable for the detection of Lactobacillus plantarum in fermented milk.

A quality control sample for the qualitative analysis of Listeria monocytogenes was developed by vacuum freezing drying technology, and the homogeneity and stability of the quality control sample were systematically analyzed. By optimizing the type of lyophilized matrix, the optimal conditions for the development of quality control samples were obtained. The homogeneity and stability of the developed quality control sample were verified by counting the number of cells as well as using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The quality control sample was white in color, spherical in shape and uniform in size. The results of cell counting for uniformity validation showed F = 0.567, which was less than the critical value, indicating good uniformity. In the transportation stability test, the quality control sample remained stable at 37 and 25 ℃. In the storage stability test, the resurrection rate of the quality control sample was 101.5% after 28 days of storage at –20 ℃, and 99.6% after 28 days of storage at 4 ℃, indicating that the sample has good uniformity and stability, and can be used as a positive quality control sample for the detection and quality control of Listeria monocytogenes.

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.