An Evaluation of the Sensitivity and Applicability of a Droplet Digital Polymerase Chain Reaction Assay to Simultaneously Detect Pseudomonas aeruginosa and Pseudomonas fragi in Foods

• Published in: Foods Vol. 13; no. 10; p. 1453
• Main Authors: Huang, Ju, Zhai, Ligong, Wang, Junyin, Sun, Xiaotian, Wang, Baoshi, Wei, Zhaohui
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 08-05-2024
• Subjects: Accuracy; Bacteria; Biofilms; Contamination; Disinfection & disinfectants; Drinking water; droplet digital PCR; Droplets; Drug resistance; Enrichment media; Experiments; Food; Food contamination; Food contamination & poisoning; Food spoilage; Genomes; Genomics; High temperature; Methods; Microbial contamination; Microorganisms; Milk; Pollution control; Polymerase chain reaction; Pseudomonas aeruginosa; Pseudomonas fragi; Sensitivity analysis; simultaneous detection; Spoilage; Target detection; Anhui China; China; Pseudomonas aeruginosa; simultaneous detection; Pseudomonas fragi; droplet digital PCR
• ISSN: 2304-8158; 2304-8158
• DOI: 10.3390/foods13101453

Achieving effective control over microbial contamination necessitates the precise and concurrent identification of numerous pathogens. As a common bacterium in the environment,
is rich in variety. It not only has pathogenic strains, but also spoilage bacteria that cause food spoilage. In this research, we devised a remarkably sensitive duplex droplet digital PCR (dddPCR) reaction system to simultaneously detect pathogenic
(
) and spoilage
(
). By employing comparative genomics, we identified four genes of P. fragi. Through a specific analysis, the
gene was selected as the detection target for
, and the
gene was chosen for
, which were applied to construct a dddPCR reaction. In terms of specificity, sensitivity and anti-interference ability, the constructed dddPCR detection system was verified and analyzed. The assay showed excellent sensitivity and applicability, as evidenced by a limit of detection of 10
cfu/mL. When the concentration of natural background bacteria in milk or fresh meat was 100 times that of the target detection bacteria, the method was still capable of completing the absolute quantification. In the simulation of actual sample contamination,
could be detected after 3 h of enrichment culture, and
could be detected after 6 h. The established dddPCR detection system exhibits exceptional performance, serving as a foundation for the simultaneous detection of various pathogenic bacteria in food products.