Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor

doi:10.13560/j.cnki.biotech.bull.1985.2023-0698

Abstract

Abstract:

Escherichia coli O157:H7 is an important food borne pathogen related to public health. Antibody molecules are the most important effector molecules in the humoral immune response. They have a variety of biological activities. The main biological function is to specifically bind to the corresponding antigen. Aptamers are shorter DNA sequences synthesized in vitro, which can bind specifically by recognizing specific regions and targets. Based on the specific recognition function of antibody and aptamer, the capture effect and magnetic separation of immunomagnetic beads, and the construction of biosensor by polyclonal antibody and tailoring aptamer, real-time fluorescence quantitative polymerase chain reaction（qPCR）may achieve the detection of target at (8×10³)-(8×10⁶) CFU/mL. The limit of detection is 800 CFU/mL for quantitative detection.

Key words: polyclonal antibody, aptamer, tailoring, immunomagnetic beads, Escherichia coli O157: H7

HOU Wei-chen, YE Ke, LI Jie, ZHANG Yang-zi, XU Wen-tao, ZHU Long-jiao, LI Xiang-yang. Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor[J]. Biotechnology Bulletin, 2023, 39(12): 81-89.

Figures/Tables 10

Table 1 Nucleic acid sequences used in the experiment

名称 Name	序列 Sequence（5'-3'）
S1	CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT ATG GGT GGT GGA TGA GTG TGT GGC TCG CAC TGC TCC TGA ACG TAC
S1-1	CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT A TCG CAC TGC TCC TGA ACG TAC
S1-2	CAG GTC CAT CGA GTG GTA GGA TGG GTG GTG GAT GAG TGT GTG GC TCG CAC TGC TCC TGA ACG TAC
S1-3	CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TGG GTG GT TCG CAC TGC TCC TGA ACG TAC
S1-3-1	CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TCG CAC TGC TCC TGA ACG TAC
FP	CAG GTC CAT CGA GTG GTA GGA
RP	GTA CGT TCA GGA GCA GTG CGA

Fig. 1 Schematic diagram of antibody aptamer sandwich method for detecting Escherichia coli

Fig. 2 Feasibility analysis of adaptor clipping A：Amplification curve of original bacterial solution adaptor and gradient dilution.B：Change trend of original bacterial solution adaptor and gradient dilution

Fig. 3 Comparison of tailored adaptor and original adaptor A: S1 and S1-1 amplification curves; B: S1 and S1-2 amplification curves; C: S1 and S1-3 amplification curves; D: S1 and S1-3-1 amplification curves

Fig. 4 Characterization of IMB A: Particle size change; B: potential change

Fig. 5 Feasibility analysis for biosensor A: Amplification curves of three complexes of IMB target adaptor, IMB adaptor, and IMB target. B: Amplification curves after four times ten fold gradient dilution of bacterial solution

Fig. 6 Condition optimization results A: Optimization of the type of adapted body scissor binding buffer; B: optimization of the pH of adapted body scissor buffer; C: optimization of adapted body scissor incubation temperature; D: optimization of sensor IMB target incubation time

Fig. 7 Sensitivity measurement

Fig. 8 Specificity of detection method for E. coli O157∶H7 A: Tailor adaptor specificity; B&C: interspecific specificity of sensors

Table 2 Actual sample testing

样品 Sample	传统培养法Traditional cultivation method /（CFU·mL^-1）	检测量Detection quantity/ （CFU·mL^-1）	回收率 Rate of recovery/%	RSD/%
1	3×10³	2.89×10³	96.3	2.15
2	3×10⁴	3.11×10⁴	103	2.07
3	3×10⁵	3.26×10⁵	108	1.35

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