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MicroRNA detection method based on Hg<2+> assistance and double-polymerase isothermal nucleic acid amplification technology

A technology of isothermal nucleic acid amplification and detection method, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effect of sensitive detection

Active Publication Date: 2016-11-16
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SYBR Green І has good optical properties, temperature stability and sensitivity. It can be inserted into DNA double strands and emits a strong fluorescent signal after combining with DNA double strands. However, it also has certain non-specific adsorption to single-stranded nucleic acids.

Method used

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  • MicroRNA detection method based on Hg&lt;2+&gt; assistance and double-polymerase isothermal nucleic acid amplification technology
  • MicroRNA detection method based on Hg&lt;2+&gt; assistance and double-polymerase isothermal nucleic acid amplification technology
  • MicroRNA detection method based on Hg&lt;2+&gt; assistance and double-polymerase isothermal nucleic acid amplification technology

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Experimental program
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Effect test

Embodiment 1

[0019] (1) Mix microRNA and template DNA (cpDNA), anneal and hybridize at 80 °C for 5 min, then slowly cool to room temperature, and add polymerase-containing Klenow fragment (KFexo - ), deoxythymidine triphosphate and terminal deoxynucleotidyl transferase (TdTase) in 20 mM pH 7.9 with 50 mM KAc, 10 mM Mg(Ac) 2 and 0.25 mM CoCl 2 tris acetate buffer solution, reacted in 37 ° C water bath for 3 h, made polythymine base sequence, added Hg 2+ After mixing completely, react isothermally in a water bath at 37 °C for 1 h to form T-Hg 2+ -T double-strand structure solution;

[0020] (2) Add 5 μL SYBR Green І to T-Hg 2+ -T double-strand structure solution, mixed and transferred to a centrifuge tube, with 10mM pH 7.5 containing 50mM NaCl and 10mM MgCl 2 Tris hydrochloride buffer solution was diluted to 200 μL, reacted at room temperature for 10 min, in a quartz cell with an optical path length of 1 cm, when the excitation wavelength was 490 nm and the scanning range was 510-650 nm,...

Embodiment 2

[0023] The effect of two polymerases on the reaction during polymerization was studied by fluorescence spectroscopy and polyacrylamide gel electrophoresis. Depend on image 3 It can be seen that in the cpDNA+microRNA-21 solution, only when the polymerase KFexo - In the presence of TdTase and TdTase, the fluorescence signal of SYBRGreen І was significantly enhanced (curve a), while only one of the polymerases or no polymerase, only a very weak fluorescence signal (curve b-e), the above results show that the amplification of the fluorescence signal Depends on polymerase KFexo - and TdTase co-action. Polyacrylamide gel electrophoresis analysis of reaction products of double polymerase extension thymine (inset), when only polymerase TdTase or KFexo - When , no amplification product was generated (bands 1-4); while when the polymerase TdTase and KFexo - When added at the same time (lane 5), a large amount of amplification product was generated, which was so long that it remained ...

Embodiment 3

[0025] Figure 4 is the change of SYBR Green І fluorescence intensity with the concentration of microRNA-21. The fluorescence intensity of SYBR Green І increases with the increase of the concentration of microRNA-21. The concentration of microRNA-21 is in the range of 10 pM-10 nM. The fluorescence intensity of SYBR Green І has a good linear relationship with the logarithm of the concentration of microRNA-21 (inset ), the detection limit of microRNA-21 was 5 pM.

[0026] In order to evaluate the specificity of this method for the detection of microRNA-21, a control experiment was carried out: microRNA-210, microRNA-141 and single base mismatch (SM) microRNA-21 were used as negative controls, and samples without microRNA-21 were Blank control (Blank), the results are as follows Figure 5 shown. In the presence of MicroRNA-210 and microRNA-141, the fluorescence intensity of SYBR Green І was similar to that of the blank control; in the presence of single base mismatched microRN...

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Abstract

The invention discloses a microRNA detection method based on Hg<2+> assistance and a double-polymerase isothermal nucleic acid amplification technology, and belongs to the technical field of optical bio-sensing. On the basis of a combined catalytic action of a polymerase Klenow fragment and terminal deoxynucleotidyl transferase, microRNA is effectively extended into a polythymine base sequence which is relatively long; Hg<2+> is added, so that a T-Hg<2+>-T double-stranded structure is formed; an SYBR Green I fluorescent dye is interpolated into the T-Hg<2+>-T double-stranded structure, so that the fluorescence of the SYBR Green I is enhanced; and a fluorescence enhancing degree is positively related to the concentration of the microRNA; therefore, the sensitive detection of the microRNA is achieved.

Description

technical field [0001] The present invention relates to a kind of based on Hg 2+ The invention relates to a microRNA detection method of auxiliary and double polymerase isothermal nucleic acid amplification technology, which belongs to the field of optical biosensing technology. Background technique [0002] MicroRNA is a class of endogenous non-protein-coding RNA molecules with a length of 19-23 bases, which play a pivotal role in many biological processes. MicroRNA in organisms can regulate protein expression by inhibiting translation or degrading target messenger ribonucleic acid molecules. In recent years, studies have found that changes in the expression level of microRNA are closely related to the type of cancer, the stage of development and even the efficacy of drugs. Therefore, it is of great significance to explore new methods for microRNA detection with high sensitivity and good selectivity. Northern blotting and microarray techniques are commonly used methods f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2525/207C12Q2563/137C12Q2521/131C12Q2563/107
Inventor 梁汝萍迟宝珠邱建丁
Owner NANCHANG UNIV
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