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Nucleic acid aptamer for combining cholera enterotoxin subunit B and application thereof

A nucleic acid aptamer, enterotoxin technology, applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc. Obtain the effect of simple, fast, low price and good application prospect

Inactive Publication Date: 2015-03-04
珠海国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional Vibrio cholerae method needs to be isolated first, then cultivated, and then identified by the classic method. Time-consuming and insensitive are the common problems of these methods
Immunological methods are simple, convenient, rapid, and specific, but there are still shortcomings such as serious cross-reaction, many false positives, and low sensitivity.
Although the polymerase chain reaction (PCR) technology has the characteristics of accuracy, sensitivity, and rapidity, it is complicated to operate when detecting a large number of samples, so it is derived on the basis of the polymerase chain reaction (PCR) technology. Many new polymerase chain reaction (PCR) technologies, such as multiplex PCR technology, although multiplex PCR simplifies the operation of PCR experiments, but because this technology requires several pairs of primers to amplify at the same time, it is easy to produce non-specific bands or false positives. Positive, affecting test results
However, there is no report specifically for the detection of cholera enterotoxin

Method used

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  • Nucleic acid aptamer for combining cholera enterotoxin subunit B and application thereof
  • Nucleic acid aptamer for combining cholera enterotoxin subunit B and application thereof
  • Nucleic acid aptamer for combining cholera enterotoxin subunit B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Screening of a single-stranded DNA aptamer capable of binding to cholera enterotoxin B subunit

[0016] Using SELEX technology, proceed as follows:

[0017] 1. Synthesis of ssDNA random library and primers

[0018] The total length of the ssDNA library used for SELEX screening is 81nt, in which both ends are fixed sequences bound by primers, and the middle 35 nucleotides are random sequences.

[0019] The library sequences are:

[0020] 5'-CCATGCACTGGTAGTCTGTCGAAGT-(N35)-AGATAGCTGAATGTCCGCCTA-3'.

[0021] The primer sequences for the amplified library were:

[0022] Upstream primer: 5'-CCATGCACTGGTAGTCTGTCGAAGT-3';

[0023] Downstream primer: 5'-TAGGCGGACATTCAGCTATCT-3';

[0024] Biotin-labeled upstream primer: 5’-Biotin-CCATGCACTGGTAGTCTGTCGAAGT-3’

[0025] The random ssDNA library and primers were synthesized by Bao Biological Engineering (Dalian) Co., Ltd. and purified by PAGE.

[0026] 2. SELEX screening process of cholera enterotoxin B subunit apta...

Embodiment 2

[0036] The ssDNA library of embodiment 2 screening and the mensuration of target protein binding rate

[0037] (1) Asymmetric PCR amplification was performed using biotin-labeled upstream primers and ordinary downstream primers to obtain a biotin-labeled ssDNA library at the 5' end. After electrophoresis, the gel was cut and purified, and the concentration was adjusted to 1.5 pmol / μl.

[0038] (2) Coating a 96-well ELISA plate with cholera enterotoxin B subunit protein, 200 ng per well, and setting blank control wells in parallel.

[0039] (3) After high-temperature denaturation at 95°C for 5 minutes, add the labeled ssDNA library of each round to the reaction well for binding reaction with the toxin protein, incubate at 37°C for 2 hours, and discard the liquid in the well.

[0040] (4) wash 3 times with washing buffer (PBS+0.05% Tween20, PBS-T, pH7.4), each 3min, then add freshly prepared streptavidin-horseradish peroxidase conjugate ( HRP-labeled Streptavidin, diluted 1:100...

Embodiment 3

[0044] Example 3 Detection of cholera enterotoxin B subunit by membrane hybridization method using biotin-modified nucleic acid aptamer

[0045] The affinity between the aptamer and cholera enterotoxin B subunit was determined by membrane hybridization. First, re-synthesize the aptamer described in SEQ ID No.1 and label it with biotin at the 5' end, dissolve it with coating buffer (0.05mol / L NaHCO3, pH9.6) and make it 1μM; cut 1 sheet 1.6×3.8cm nitrocellulose membrane strips, drawn into 6 grids with seal oil; numbered 1-6, of which grid 1 is the negative control of BSA (0.5 μg), grid 2 is the blank control (without cholera enterotoxin protein ); the sample volumes of No. 3, 4, 5, and 6 cholera enterotoxin B subunits were: 0.05, 0.1, 0.2, and 0.5 μg, and the sample volume was 1.5 μL. Place the membrane strips in a small square box, add 2ml blocking solution (10% skimmed milk powder) and carry out blocking treatment at room temperature for 2 hours; then wash 2 times with PBS, t...

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Abstract

The invention relates to an oligonucleotide aptamer capable of specifically combining a cholera enterotoxin subunit B and an application thereof. The aptamer is a single-chain DNA molecule with 81 base lengths, and has a nucleotide sequence described in a sequence table SEQ ID No. 1. The aptamer disclosed by the invention is screened by adopting a system evolution index enrichment technology, can be used for preparing a detection reagent of cholera enterotoxin, and has the advantages of stability, simplicity and convenience, high speed, economic performance and the like.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a nucleic acid aptamer capable of specifically binding to cholera enterotoxin B subunit with high affinity and its application in the detection of cholera enterotoxin. Background technique [0002] Cholera is a severe intestinal infectious disease caused by Vibrio cholerae infection of O1 group and O139 group. It is one of the international quarantine infectious diseases characterized by acute onset, rapid spread, wide spread and the ability to cause a pandemic. . my country has listed cholera as a class A legal infectious disease, and it is an infectious disease that should be quarantined at border ports. Vibrio cholerae mainly causes clinical symptoms such as severe diarrhea and vomiting through the toxin it secretes. Cholera enterotoxin (CT) is the most important exotoxin in Vibrio cholerae, and it is also the main pathogenic factor causing cholera. It consis...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/68
CPCY02A50/30
Inventor 莫秋华谭华汪海波冯子力杨泽林继灿
Owner 珠海国际旅行卫生保健中心
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