PCR couple with magnetic nanoparticles-based biotin-avidin system for chemiluminescent detection of specific gene (rfbE) of E. coli O157

摘要

A novel method was established through the amplification of chemiluminescent signals of nucleic acid hybridization based on magnetic nanoparticles (MNPs) and biotin-avidin system. Amino-modified specific probe was firstly coupled to MNPs with carboxylic acid. Then the probes on the MNPs were hybridized with the biotin-dUTP-labeled DNA fragments obtained by PCR amplification. Streptavidin-modified alkaline phosphatase was applied to bind with the biotin, and the alkaline phosphatase reacted with 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane to generate a light-emitting product. Specific gene (rfbE) of E. coli O157 antigen was studied to evaluate this system. The MNPs immobilized with specific probe of rfbE could specifically capture the biotin-labeled DNA fragment of rfbE rather than that of the chloramphenicol acetyltrans-ferase (CAT) gene.