| Escherichia coli O157:H7(E.coli O157:H7),a type of enterohemorrhagic E.coli,is a highly pathogenic food–borne pathogen.The infectious dose of E.coli O157:H7 is estimated to be as low as 10 viable cells and the detection of E.coli O157:H7 has attracted considerable attention.The traditional sandwich enzyme linked immunosorbent assay(ELISA),based on biotin–streptavidin signal amplification ELISA,based on HCR and biotin–streptavidin signal amplification ELISA were studied to establish an accurate,sensitive,and rapid detection method of E.coli O157:H7.In this paper,the traditional sandwich ELISA method was firstly studied to detect E.coli O157:H7.Under optimal mAb concentrations,the limit of detection of the traditional ELISA in pure culture and in whole milk was 4.88 × 104 CFU/mL and 5.08 × 104 CFU/m L,respectively.The coefficient of variation of the traditional ELISA in pure culture and in whole milk was 1.31%–6.87% and 1.52%–7.81%,respectively.The biotin–streptavidin signal amplification ELISA method was studied for detecting E.coli O157:H7.Under optimal bio–mAb conccentrations,the limit of detection in pure culture and in whole milk was 2.00 × 104 CFU/m L and 3.23 × 104 CFU/m L,respectively.The coefficient of variation of the biotin–streptavidin signal amplification ELISA in pure culture and in whole milk was 1.12%–8.63% and 1.25%–9.61%,respectively.The traditional sandwich ELISA and the biotin–streptavidin signal amplification ELISA were simple,rapid,specificity,and could be applied in the detection of whole milk samples inoculated with E.coli O157:H7.But the sensitivity of both methods were poor.A novel sandwich ELISA was developed for the sensitive determination of E.coli O157:H7 by using DNA–based hybridization chain reaction(HCR)and biotin–streptavidin signal amplification.The anti–E.coli O157:H7 polyclonal antibody(pAb)was immobilized in the ELISA wells.The anti–E.coli O157:H7 monoclonal antibody(mAb)and initiator strand(DNA1)were labeled on gold nanoparticle(Au NP)to form a mAb–AuNP–DNA1 complex.In the presence of the target E.coli O157:H7,pAb–E.coli O157:H7–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.In this study,the key parameters included the concentrations of mAb(150 ?g/mL),pAb(10 ?g/m L),bio–H1 and bio–H2(0.25 nmol/m L),the amount of DNA1(400 ?L;1 nmol/mL),the choice of blocking agent(3% BSA),the HCR reaction time(75 min),and the chromogenic reaction procedure(incubation time 10 min;temperature 37 °C)?Under optimal conditions,E.coli O157:H7 could be detected in the range of 5 × 102 CFU/m L to 1 × 107 CFU/m L;the limit of detection in pure culture and in whole milk was 1.08 × 102 CFU/mL and 2.60 × 102 CFU/m L,respectively.The proposed method is considerably specific,and the coefficient of variation of in pure culture and in whole milk was 0.99%–5.88% and 0.76%–5.38%,respectively.The LOD of the novel ELISA were 451 times and 185 times lower than that of traditional sandwich ELISA and biotin–streptavidin signal amplification ELISA,respectively.The method is simple,rapid,sensitive,and demonstrates good specificity for E.coli O157:H7 detection.These features demonstrate that the promising novel ELISA methods could be used for the rapid detection of low levels of E.coli O157:H7. |
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