Detection of 13 foodborne pathogens in aquatic products using visual chromogenic chips based on asymmetric multiplex polymerase chain reaction and nucleic acid hybridization

The ability to efficiently and accurately detect pathogenic microorganisms in food is a critical tool for ensuring food safety. However, traditional culture and PCR techniques, regarded as the gold standard, constantly face shortcomings. To overcome these limitations, we developed a technique utilizing multiplex asymmetric PCR (MAPCR) with a chromogenic DNA microarray to detect 13 pathogenic microorganisms. Primers and probes were designed based on 13 specific genes, and single strands were enriched through asymmetric PCR. These single strands then hybridized with the corresponding probes on the chip, which were finally colored using a biotin-streptavidin-alkaline phosphatase indicator system. Our results showed that for individual tests, the limit of detection (LOD) was found to be: Y. enterocolitica, P. putida, V. mimicus, V. alginolyticus, S. flexneri, C. sakazakii, E. coli O157: H7, S. pyogenes, V. parahaemolyticus, L. monocytogenes, and V. vulnificus are all 10 pg/μL; S. enterica and V. cholerae are 100 pg/μL. Additionally, the multiplex assay exhibited excellent sensitivity at 10 pg/μL along with good specificity. This technique also obtained LOD values between 104-105 CFU/25 g in tests of food sample testing. Finally, this assay has good stability and preservation capability. In summary, this study presents an efficient method that can accurately identify multiple pathogens simultaneously, making it an attractive option for use in food safety testing.