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A group of oligonucleotide aptamers that specifically recognize Escherichia coli O157:H7 in different growth stages

An oligonucleotide and Escherichia coli technology, which is applied in the field of food safety biology, can solve the problems such as the inability to meet the detection requirements, and achieve the effects of easy labeling, stable properties and convenient synthesis.

Active Publication Date: 2021-03-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the aptamer can only specifically recognize E. coli in the logarithmic phase, and the aptamer dissociation constant K d It is 82.45±18.46nM, and the detection limit is 90cfu / mL, which cannot meet the detection requirements

Method used

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  • A group of oligonucleotide aptamers that specifically recognize Escherichia coli O157:H7 in different growth stages
  • A group of oligonucleotide aptamers that specifically recognize Escherichia coli O157:H7 in different growth stages
  • A group of oligonucleotide aptamers that specifically recognize Escherichia coli O157:H7 in different growth stages

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Screening of aptamers

[0026] 1. Synthesize random single-stranded DNA library and primers (completed by American Industrial Integrated DNA Technologies)

[0027] Random ssDNA library:

[0028] 5’-TGA GCC CAA GCC CTG GTA TG-N40-GGC AGG TCT ACT TTG GGA TC-3’

[0029] 5'-carboxyfluorescein-labeled upstream primer: 5'-FAM-TGA GCC CAA GCC CTG GTA TG-3'

[0030] 5' phosphorylation downstream primer: 5'-P-GAT CCC AAA GTA GAC CTG CC-3'

[0031] The random ssDNA library and primers were prepared with TE buffer as a 100uM stock solution and stored at -20°C for future use.

[0032] 2. Selection and processing of bacteria

[0033] (1) Determination of the growth curve of Escherichia coli O157:H7

[0034] Escherichia coli O157:H7 was cultured in LB (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH7.4) liquid medium, 37°C, 120r / min shaker culture, The OD value of the bacterial solution was measured at regular intervals. According to the value of each ...

Embodiment 2

[0047] Example 2: Aptamer affinity and specificity testing

[0048] (1) Determination of dissociation constant

[0049] The synthesized 9 aptamers were diluted with TE buffer to prepare a 10 pmol / L solution, and stored at -20°C for later use. The affinity of 9 aptamers was analyzed by BDFACS Calibur flow cytometer. Different volumes of 10 pmol / L aptamers were added to 500 μL of BB binding buffer and diluted to different concentration gradients (10, 50, 100, 150, 200 nmol / L), denatured at 95 °C for 10 min, and immediately cooled at 0 °C for 10 min. The aptamer solution was added to the treated E. coli O157:H7 and incubated at 37°C with slow shaking for 1 h.

[0050] It was then washed with BB buffer and resuspended in 500 μL BB buffer for flow cytometry analysis. In the flow cytometry analysis, the fluorescence intensity of the blank sample (without aptamer) was adjusted first, and then the forward scatter, side scatter and fluorescence intensity of the sample were measured ...

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Abstract

The invention discloses a group of oligonucleotide aptamer capable of specifically recognizing escherichia coli O157:H7 in different growth periods, and belongs to the technical field of food safety biology. A whole cell-SELEX technology is used; the escherichia coli O157:H7 in different growth periods are used as targets; through 14 rounds of repeated screening, appetency and specificity test analysis validation, a group of oligonucleotide aptamer with good appetency and specificity by aiming at escherichia coli O157:H7 in different growth periods is obtained. The aptamer has wide applicationprospects in the aspect of accurately, fast and sensitively detecting the escherichia coli O157:H7 in food.

Description

technical field [0001] The invention relates to the field of food safety biotechnology, in particular to a method for screening oligosaccharides capable of specifically recognizing Escherichia coli O157:H7 in different growth stages by using SELEX technology in molecular biology technology (that is, exponential enrichment ligand system evolution technology). Nucleotide aptamers. Background technique [0002] Escherichia coli O157:H7 (Escherichia coli O157:H7, E. coli O157:H7) is the main pathogenic strain of Enteroheamorrhagic Escherichia coli (EHEC) pathogenic Escherichia coli. It is a spore-free, flagellated, gram-negative bacillus with strong acid resistance and low temperature resistance. The main mode of transmission is food contaminated with feces. [0003] Escherichia coli 0157:H7 is extremely virulent and can be infected by less than 50 pathogenic bacteria, showing symptoms such as hemorrhagic enteritis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/569
CPCC12N15/115C12N2310/16G01N33/56916G01N2333/245
Inventor 王周平邹颖段诺
Owner JIANGNAN UNIV
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