| ABSTRACT:ObjectiveTo establish a high-throughput and rapid detection method based on Luminex xMAP suspension array technology for simultaneous detection of seven important foodborne pathogens, including Vibrio parahaemolyticus,Vibriocholerae,Salmonella,Staphylococcusaureus,Escherichiacoli O157,Listeria monocytogenes and Campylobacter jejuni.MethodsSpecific genes of the seven 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 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. ResultsAll7 kinds of target pathogens showed specific amplification without cross-reaction with non-target strains.Double log linear regression analysis of MFI values of the seven pathogens at concentrations ranging from 101 to 10? CFU/mL,showed an extremely significant positive correlation between bacterial concentration and MFI value (R2>0.994, P<0.001), with a slight species-specific slope.The minimum quantitative detection limit was 102 CFU/mL,and the minimum qualitative detection limit was close to 101CFU/mL.The intra-assay coefficient of Variation CV% of the seven 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 96.51%.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.ConclusionsThe 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 seven foodborne pathogens,and is suitable for food safety monitoring,emergency disposal of food poisoning and rapid clinical detection. |
