Highly Selective, Sensitive and Rapid Detection of Escherichia coli O157: H7 Using Duplex PCR and Magnetic Nanoparticle-Based Chemiluminescence Assay

摘要

A novel strategy based on duplex PCR and chemiluminescence (CL) was developed in this study to identify Escherichia coli (E. coli) O157: H7 serotype. The antigen epitopes O157 gene (rfbE) and flagella antigen H7 gene (fliC) were simultaneously amplified and labelled with biotin through duplex PCR. The biotinylated rfbE and fliC amplicons were captured to the surface of oligonucleotide probe-modified magnetic nanoparticles (MNPs) successively. After being conjugated with alkaline phosphatase-labelled streptavidin (ALP-STA), the substrate 3-(2'-Spiroadamantane)-4-methoxy-4( 3''-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) was mixed with the magnetic complexes and CL signals were generated immediately. The stable CL intensity reflected the amount of rfbE and fliC genes, which confirmed the presence of E. coli O157: H7. The detection limit was 10 pM under optimized conditions. The detection results from food samples indicated that this approach is reliable and repeatable, thus holding great potential for rapid, highly selective and sensitive food inspection.