Application of Biofunctionalized Nanoparticles for Detection of Escherichia Coli O157:H7
|Keywords||Magnetic nanoparticles Gold nanoparticles Bio-barcode PCR DNA probes E. coli O157: H7|
E. coli O157: H7 (Escherichia coli O157: H7, E. coli O157: H7) is a major enterohemorrhagic Escherichia coli serotype had several outbreaks worldwide, a serious threat to the health of people and caused huge economic losses. Established two bio-functionalized nanoparticles for detection of E. coli 0157: H7, and for the detection of the artificially contaminated milk samples, the main research is as follows: Preparation of bio-functionalized nanoparticles. Using sodium citrate reduction prepared particle diameter of 13 nm gold nanoparticles (Au-NPs), polyclonal antibodies with E. coli O157: H7, thiolated capture DNA chain, barcode DNA strand, DNA probes, chains, etc. Preparation of biological functions of Au-NPs, the use of UV-vis, TEM preparation and modification process characterization. By coprecipitation prepared particle size of 27 ± 4 nm magnetic nanoparticles (MNPs), 3 - aminopropyl triethoxysilane (APTES) the modification and glutaraldehyde cross-linking was prepared by E. coli 0157: H7 monoclonal antibody functionalized MNPs. Bio-functionalized nanoparticles detection of E. coli 0157: H7 studied. Use of bio-functionalized MNPs and Au-NPs target the immune response of E. coli 0157: H7, the formation of the the MNPs / target / Au-NPs \the barcode DNA release quantitative analysis of E. coli 0157: H7, by PCR amplification and electrophoresis, the establishment of a bio-barcode of the conventional PCR-based detection methods. The minimum detection limit of the method of standard samples 10CFU/mL, detection range 10-106CFU/mL; with other E. coli serotypes common foodborne pathogens such as E. coli 086: H2, E. coli 0111: K58, E. coli K12, Salmonella are not cross-react, specificity; better reproducibility of the method, the average coefficient of variation of the intra-reproducibility as (6.6 ± 3.3)%, (7.3 ± 3.6)% of the average coefficient of variation of the inter-assay reproducibility. The former on the basis of a method, the barcode marked on the Au-NPs DNA release, using DNA probe hybridization barcode DNA causing AU-NPS color change create based on bio-barcode probe and method for the detection of the Au-NPs, the results show that can reach the lowest detection limit of the standard sample 102CFU/mL, this method has good specificity; repetitive experiments, the average coefficient of variation of the within-batch and batch-to-batch reproducibility were (2.9 ± 1.3)% and (3.2 ± 1.5)%. E. coli O157: H7 artificially contaminated milk sample detection research. Application of conventional PCR-based bio-barcode artificially contaminated by E. coli O157: H7 milk sample detection limit of detection for 10CFU/mL; minimum detection limit of the sample measured based bio-barcode probe and the detection method of the Au-NPs 102CFU/mL and compare the two detection methods.