AgNCs/HpDNA probe based microRNA SDA (strand-displacement amplification) detection method

A detection method, mir-19b-3p technology, applied in DNA/RNA fragment, recombinant DNA technology, microbial assay/inspection, etc., can solve problems such as difficulty in ensuring specificity, shorten reaction time and save reaction materials Effect

Inactive Publication Date: 2016-04-13
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although simple hybridization detection is

Method used

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  • AgNCs/HpDNA probe based microRNA SDA (strand-displacement amplification) detection method
  • AgNCs/HpDNA probe based microRNA SDA (strand-displacement amplification) detection method
  • AgNCs/HpDNA probe based microRNA SDA (strand-displacement amplification) detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Feasibility verification of the detection scheme

[0053] (1) AgNCs / HpDNAs probe synthesis

[0054] The synthesis was carried out according to the method in Yeh's article. HpDNAs(GRE19b(5s)C), AgNO 3 , NaBH 4 The starting concentrations are 100 μM, 1 mM and 1 mM, respectively. The reserve concentration of phosphate buffer is 200mM (Pi, pH8.0). Equimolar AgNO 3 And NaBH 4 Follow 1GRE19b(5s)C:17AgNO in turn 3 :17NaBH 4 Was added to HpDNA to make the three final concentrations 15μM, 250μM and 250μM (Pi, 20mM, pH8.0). Among them, NaBH 4 Need fresh configuration, and finally quickly added to Ag within 30s + / HpDNA mixture, after that, shake vigorously for 45s~1min. The resulting solution was placed in a dark environment at room temperature for 18 hours to obtain a stable AgNCs / HpDNAs probe.

[0055] (2) Verification of fluorescence enhancement effect of G-rich sequence hybridization

[0056] To the obtained probe AgNCs / GRE19b(5s)C, respectively add a certain amount o...

Embodiment 2

[0059] Example 2. Detection of a single miRNA

[0060] (1) AgNCs / GRE19b(5s) C probe synthesis

[0061] Prepare AgNCs / GRE19b(5s)C probe according to the steps described in (1), but the final concentration of GRE19b(5s)C is 5μM, AgNO 3 And NaBH 4 The amount added is still 1GRE19b(5s)C:17AgNO 3 :17NaBH 4 get on.

[0062] (2) Single miRNA detection

[0063] Each reaction tube contains 50μLSDA reaction solution, which contains the following components: 1×Nb2.1 homemade buffer (buffer pH 7.925℃) (50mMNaAc, 10mMTris-HAc, 10mMMg(Ac) 2 And 100μg / mLBSA) 200μM dNTPs, 10UBsu polymerase (no DTT), AgNCs / GRE19b(5s) C probe (2.5μMHpDNA), different concentrations of target miRNA (0.05-2.5μMmiR-19b-3p) and 2.5μM primer Pri6( 7s). The resulting reaction solution was incubated at 55°C for 55 minutes, and then stored in a dark environment at 4°C, and then fluorescence detection could be performed on a fluorescence spectrophotometer. The experiment was repeated 3 times. In addition, the reaction solutio...

Embodiment 3

[0068] Example 3. Single-base mismatch nucleic acid detection

[0069] (1) Single-base mismatch nucleic acid detection

[0070] Follow the steps described in (1, 2) in Example 2, only change the nucleic acid to be tested to 0.5μM mismatch nucleic acid, that is, detect miR-16-5p, h19bDMⅠ (sequence SEQIDNO) based on AgNCs / GRE19b(5s)C probe : 5) and h19bDMII (sequence SEQ ID NO: 6).

[0071] (2) Results

[0072] The results of mismatch nucleic acid detection are as Figure 5 Shown. It can be seen that in the detection based on AgNCs / GRE19b(5s)C probe, only miR-19b-3p (λex=490nm) showed the highest fluorescence enhancement signal, and the fluorescence enhancement of the remaining samples at this excitation wavelength was relatively higher. Weak, in addition, based on AgNCs / GRE19b(5s)C probe excited at 430nm wavelength, miR-19b-3p and h19bDMI also exhibited fluorescence quenching, while miR-16-5p and h19bDMI exhibited opposite fluorescence enhancement. In summary, the detection based ...

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Abstract

The invention discloses an AgNCs/HpDNA probe based microRNA SDA (strand-displacement amplification) detection method. The method includes that silver nanoclusters are synthesized by the aid of a hairpin type DNA template and serve as novel molecular bacons, and single detection of gastric plasm miRNA markers is realized by a hybridization resulted G-rich fluorescence enhancement effect in strand displacement isothermal amplification reaction mediated by a G-rich sequence of a dangling end of a primer. The AgNCs/HpDNA probe based microRNA SDA detection method has the advantages of high specificity, short reaction time, low material consumption and simplicity and convenience in operation and creates a new direction for establishing a quick and simple novel miRNA detection method.

Description

Technical field [0001] The invention relates to the field of medicine and molecular diagnosis. It is a microRNA SDA detection method based on AgNCs / HpDNA probe. In particular, it relates to a hairpin-type DNA template-based synthesis of silver nanocluster probe binding chain to replace a single microRNA in isothermal amplification. Detection method. Background technique [0002] MicroRNA (miRNA) is a type of endogenous non-coding small RNA molecule with a length of approximately 21 nt, which can participate in regulating the expression level of mRNA through precise or imprecise complementary pairing with mRNA. Multiple miRNAs can coordinately regulate one mRNA, or one miRNA can also affect multiple target genes at the same time, thus forming a highly complex regulatory network that affects a series of biological functions from the molecular to cellular to tissue levels. Abnormal expression of miRNA is closely related to disease and cancer. What is particularly important is...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2525/207C12Q2525/301C12Q2531/119C12Q2563/107
Inventor 崔大祥张晶璞
Owner SHANGHAI JIAO TONG UNIV
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