To establish a method for simultaneous detection of 7 kinds of important foodborne pathogens, including Vibrio parahaemolyticus, Vibriocholerae, Salmonella, Staphylococcusaureus, Escherichiacoli O157, Listeria monocytogenes and Campylobacter jejuni by high-throughput and rapid detection method based on Luminex xMAP liquid-phase microarray technology.

Specific genes of the 7 kinds of pathogens, namely TLH, gspD, invA, nuc, rfbE/eae, hlyA and hipO, were selected as detection targets, and specific primers tagged with TAG sequence and biotin were designed and synthesized. The multiplex polymerase chain reaction (PCR) amplification system and suspension array hybridization conditions were optimized, and the specificity, sensitivity and repeatability of the method were systematically evaluated. A total of 172 clinically isolated strains were used for practical application verification.

All 7 kinds of target pathogens showed specific amplification without cross-reaction with non-target strains. Double log linear regression analysis of median fluorescence intensity values of the 7 kinds of pathogens at concentrations ranging from 10¹ to 10⁵ CFU/mL, showed an extremely significant positive correlation between bacterial concentration and median fluorescence intensity value (R²>0.994, P<0.001), with a slight species-specific slope. The quantitative limits of detection of PCR was 10² CFU/mL, and the qualitative limit of detection was close to 10¹ CFU/mL. The intra-assay coefficient of variation (CV) of the 7 kinds of pathogens at low, medium and high concentrations were 0.9%–4.6%, 0.4%–2.3% and 0.1%–0.8% respectively; the inter-assay CV were 1.5%–4.8%, 0.2%–1.7% and 0.1%–0.9% respectively, indicating excellent intra-assay precision and inter-assay stability.The total coincidence rate of detection for 172 clinically isolated strains was 96.5%. Modified Kappa consistency test showed that the overall Kappa coefficient was 0.928 (P<0.001), and the Kappa value of each pathogen was no less than 0.8, proving that the method had reliable positive detection consistency without systematic deviation, and could meet the accuracy requirements of clinical detection and food safety monitoring.

The Luminex xMAP liquid-phase microarray assay established in this study has the advantages of high specificity, high sensitivity, high throughput, rapidity and stability. It can realize simultaneous screening of 7 kinds of foodborne pathogens, and is suitable for food safety monitoring, emergency disposal of food poisoning and rapid clinical detection.