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Method for detecting HTLV-II DNA based on enzymatic catalysis controllable self-assembly bio-barcode

A biological barcode and self-assembly technology, applied in the field of chemiluminescence, to achieve the effects of improved detection accuracy, good technical effect, and simple operation

Active Publication Date: 2018-09-28
SHANDONG NORMAL UNIV
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Problems solved by technology

And, injecting drug users infected with HTLV-II may introduce the virus into the general population and blood donors through secondary transmission, thereby inducing global neurological disability and neuropathy

Method used

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  • Method for detecting HTLV-II DNA based on enzymatic catalysis controllable self-assembly bio-barcode
  • Method for detecting HTLV-II DNA based on enzymatic catalysis controllable self-assembly bio-barcode
  • Method for detecting HTLV-II DNA based on enzymatic catalysis controllable self-assembly bio-barcode

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preparation example Construction

[0048] In a preferred embodiment, the preparation method of magnetic microspheres in the above-mentioned step (1) comprises the following steps:

[0049] Dissolve capture probe 1 in 1×Tris-EDTA buffer to prepare a stock solution; add 10 μmol / L capture probe 1 and 10 mg / mL streptavidin-coated magnetic microspheres to ultrapure water , and incubated at room temperature for 10 min to bind the capture probe 1 to the streptavidin-coated magnetic microspheres; use magnetic separation to remove excess capture probe 1 in the solution to obtain a functionalized capture probe 1 magnetic microspheres coated with streptavidin.

[0050] The ratio of capture probe 1: magnetic microspheres: ultrapure aqueous solution is 1 μL: 1 μL: 10 μL.

[0051] Preferably, the method for preparing AuNPs in the above step (2) includes the following steps: dissolving the capture probe 2 and the reporter probe in 1×Tris-EDTA buffer to prepare a stock solution; mixing 0.5 mg / mL of AuNPs, Add 1 μmol / L captur...

Embodiment 1

[0058] Terminal deoxynucleotidyl transferase catalyzes the self-assembly of biological barcodes to achieve dendritic chemiluminescent signal amplification: terminal deoxynucleotidyl transferase needs to perform two polymerization extension reactions. In the first polymerization extension reaction, different concentrations of the target substance HTLV-II DNA was added to 20 μL hybridization solution containing freshly prepared capture probe 1 functionalized magnetic microspheres, 750 mmol / L NaCl and 75 mmol / L sodium citrate, and incubated at room temperature for 10 minutes to form 3'- Double-stranded DNA with overhanging hydroxyl ends. After magnetic separation, add 0.4 units of terminal deoxynucleotidyl transferase to 20 μL of the polymerization reaction solution composed of the separated precipitate product, including 2 μL of 100 μmol / L dTTP, 2 μL of 10× terminal deoxynucleotidyl transferase reaction buffer, 2 μL 2.5mmol / L CoCl 2 , and at 37°C, the protruding 3' terminal HTL...

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Abstract

The invention provides a chemiluminescence method for performing dendritic amplified assay on human T-cell leukemia virus II (HTLV-II) DNA based on terminal deoxynucleotidyl transferase catalysis self-assembly bio-barcode. According to the technical scheme, the method comprises the following two continuous reaction steps: (1) first, enzymatic extension and dendritic self-assembly of bio-barcode based on HTLV-II DNA induced terminal deoxynucleotidyl transferase catalysis; (2) chemiluminescence detection in presence of second enzymatic extension heme based on terminal deoxynucleotidyl transferase catalysis. According to the technical scheme in the invention, the lower limit of detection can reach 0.5*10<-18>mol / L, the sensitivity is greatly improved when being compared with that in the priorart, the specificity is good, and the operation is simple and convenient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chemiluminescent method for dendritic amplification detection of human T-lymphoblastic leukemia virus II (HTLV-II) DNA based on terminal deoxynucleotidyl transferase (TdT) catalyzed self-assembly of biological barcodes method. Background technique [0002] In the prior art, technical means commonly used to detect nucleotides include polymerase chain reaction (PCR), rolling circle amplification reaction (RCA), loop-mediated constant temperature amplification reaction (LAMP), hybridization chain reaction (HCR ), catalytic hairpin assembly (CHA) amplification, ligase chain reaction (LCR), exonuclease / endonuclease-assisted signal amplification reaction (EASA). PCR is a thermocycling-based DNA amplification technique that involves stringent primer / template design and precise thermocycling. RCA and LAMP are isothermal amplification techniques that avoid the tedious thermal cycling step...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6825G01N21/76
CPCG01N21/76C12Q1/6825C12Q1/701C12Q2563/155C12Q2563/137C12Q2521/131
Inventor 张春阳王黎娟任明
Owner SHANDONG NORMAL UNIV
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