A single-copy specific primer was designed based on beef and duck samples and through drop digital polymerase chain reaction (ddPCR) for the quantitative analysis. Results revealed that the primers had no specific amplification with sheep, chicken, pork, or other species. Both the relationships between meat weight and DNA weight and between DNA weight and DNA copy number (C) were nearly linear within the dynamic range. To calculate the original meat weight from the DNA copy number, the DNA weight was used as the intermediate value to establish the following formulae: Mbeef = 0.058C − 1.86; Mduck = 0.0268C − 7.78. To achieve a good quantitative analysis, all species used in the experiment were made of lean meat. The accuracy of the method was verified by artificial adulteration of different proportions. Testing of the commercial samples indicated that adulteration is present in the market. The established digital PCR method provided an effective tool for monitoring the adulterated meat products and reducing the adulteration in the market.
Three t ypical c ontaminant b acteria, n amely, Gluconobacter, Acetobacter, a nd Lactobacillus, w ere s elected f rom f ermented milk. The strong specific genes were screened as target sequences, and the droplet digital polymerase chain reaction (ddPCR) amplification system and reaction conditions were optimized. We established a ddPCR detection method for the three kinds of bacteria in fermented milk and then verified t he specificity an d se nsitivity of th e me thod. Ab solute qu antitative re sults were also analyzed. Results showed that ddPCR detection method for the three contaminants was specific and sensitive, and that the lowest detectable concentrations of Gluconobacter, Bacteroides, and Lactobacillus plantarum were 8.8 × 10 0 CFU/mL, 8.9 × 10 1 CFU/mL, and 9.6 × 10 1 CFU/mL, respectively. The relationship between the quantitative analysis results and ddPCR test results was good, proving the feasibility of ddPCR for absolute quantitative detection. This manuscript reports on establishment of a ddPCR detection system for three bacterial contaminants in fermented milk. The ddPCR detection system has efficient detection of targeted bacterial contaminants in fermented milks that can be valuable to the dairy industry in terms of preventing economic loss due to microbial spoilage and associated quality defects.
The trend of low breastfeeding rates increases the demand for infant milk formula (IMF) worldwide, but the use of IMF may be one of the causes of bacterial infections in infants. Complete sterility in the whole production line of IMF cannot be guaranteed; therefore, it is necessary to closely monitor the microbial content in the process. In the present study, an IMF powder production line based on the wet mixing process was sampled at 27 suspicious points in spring and summer to analyze the bacterial diversity by high-throughput sequencing. We found that 70 and 69 different bacterial phyla were present in spring and summer samples, respectively, with Proteobacteria and Firmicutes being the dominant phyla (>80% relative abundance). Moreover, 13 dominant genera each were present in spring (e. g., Pseudomonas and Lactococcus) and summer (e. g., Pseudomonas, Bacillus, and Streptococcus). Samples associated with workers showed higher bacterial species diversity (Shannon index) and richness (Chao1 index) in summer than in spring. The bacterial community composition showed high similarity between liquid milk after pasteurization and concentrated milk after evaporation. The potential bacterial pathogens were identified as Pseudomonas aeruginosa in spring and Acinetobacter baumannii in summer. Through retrospective analysis of the two opportunistic pathogens identified, it was found that the workshop environment was the potential contamination point in spring, whereas the auxiliary ingredients were the potential source of contamination in summer. The results highlight the effect of season on bacterial diversity associated with the production process of IMF and are useful in controlling the microbial quality and safety of infant dairy products.
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