DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

ABSTRACT

This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157H7 (E. coli O157H7) by using DNA-based hybridization chain reaction (HCR) and biotin-streptavidin signal amplification. The anti-E. coli O157H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb-AuNP-DNA1 complex. In the presence of the target E. coli O157H7, the sandwiched immunocomplex, which is pAb-E. coli O157H7-mAb-AuNP-DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase-streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157H7 could be detected in the range of 5×10(2) CFU/mL to 1×10(7) CFU/mL; the limit of detection was 1.08×10(2) CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157H7. The coefficient of variation of in pure culture and in whole milk was 0.99-5.88% and 0.76-5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens.