关键词: 食源性致病菌, 通用引物, 多重不对称PCR, 寡核苷酸芯片

Abstract: A new method that combined universal primer-multiplex asymmetric PCR and oligonucleotide microarray technology was developed for the simultaneous detection of seven common foodborne pathogens including Listeria monocytogenes, Salmonella, E. coli O157:H7, Staphylococcus aureus, Yersinia enterocolitica, Vibrio parahaemolyticu and a standard strain of Escherichia coli. The 5’-end of forward or reverse primer specific to each pathogen was linked with a non-homologous common sequence. Target fragments of all seven pathogens were enriched and labeled simultaneously by one-step multiplex asymmetric PCR using the Cy3-labeled common sequence as the universal primer. The labeled single-stranded amplicons were captured by the specific oligonucleotide probes immobilized on microarrays, followed by microarray scanning and analysis of fluorescence signal intensity. The results for reference strains indicated that the assay could unambiguously discriminate all seven pathogens in single and multiple infections, and it had a detection sensitivity of 0.1–1 pg of genomic DNA. Ninety-five artificially contaminated samples and retail food samples were tested by this assay, and the results agreed with those obtained with traditional culture methods and real-time PCR. This developed oligonucleotide microarray assay can provide a useful method for rapid, specific, sensitive and high-throughput detection of foodborne pathogens.

Key words: foodborne pathogens, universal primer, multiplex asymmetric PCR, oligonucleotide microarray