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Multi-nucleic acid detection method and kit

A detection kit and detection method technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long time consumption and insufficient sensitivity, and achieve the effect of short detection time, high sensitivity and multiple detection

Pending Publication Date: 2019-10-08
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Current detection methods suffer from time-consuming and insufficient sensitivity

Method used

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  • Multi-nucleic acid detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Nucleic acid sequence design of target miRNAs, DNA walkers and DNA tracks

[0043] For five targets (miRNA-29a, miRNA-151-5p, miRNA-15b, miRNA-26b, and miRNA-15a), DNA walkers (walker 1, walker 2) that can specifically hybridize to the corresponding target sequences were designed. , walker3, walker 4, and walker 5). The secondary structure of the DNA walker nucleic acid is a stem-loop structure. After forming a complementary double strand, the melting temperature of the end of the double strand is slightly lower than that of the middle region, enabling DNA respiration to occur. The DNA walker sequence consists of two parts: the target recognition region, which is used to hybridize with the target to open the stem-loop structure; the DNA track recognition region, which connects the DNA walker with the stem-loop structure opened and the DNA track (track 1, track 2, track 3 , track 4 or track 5) will be attached to the microsphere surface by hybridization. DNA track prov...

Embodiment 2

[0048] Preparation of fluorescently encoded microspheres loaded with DNA tracks

[0049] The green / red fluorescence intensity ratios of the five fluorescently encoded microspheres are 1:0, 0.1:0.1, 1:0.1, 1:1.5 and 0:1, respectively, such as figure 2 Shown in A, this figure shows flow cytometric characterization of five fluorescently encoded microspheres.

[0050] Add 50 mg / mL of EDC (1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution, stirred for 30 minutes and then added 2 μl of 100 μM 5’ amino-modified DNA orbital. After stirring and reacting at room temperature for 3 hours, add 1 ml of phosphate buffer (pH=7.4) containing 0.1% Tween-20 by mass fraction, stir for 30 minutes and then centrifuge, the precipitated microspheres are dispersed in ultrapure water, and the loaded DNA track is obtained fluorescently encoded microspheres. Connect the five DNA tracks to the corresponding fluorescently encoded microspheres accordin...

Embodiment 3

[0055] DNA walking on the surface of fluorescently encoded microspheres

[0056] 2000 fluorescently encoded microspheres of the same loaded DNA track, 20 nM of the corresponding DNA walker, 100 fM of the corresponding miRNA, 2 μl of amplification buffer, 10 mM Mg in a 20 μl reaction volume 2+ , 10mM dNTPs and 1U / μL Bst DNA polymerase, and make up the remaining volume with water (amplification buffer, Mg 2+ and Bst DNA polymerase kit purchased from NEB Beijing, Cat. No. M0537S). 0, 10, 20, 30, 40, 50 and 60 minutes were selected for the reaction at 60°C, respectively. After centrifugation and washing, excess streptavidin-modified Pacific blue fluorescent dye (purchased from Thermo Fisher Scientific, item number S11222) was added, placed at 4 degrees Celsius after sufficient shaking, and centrifuged and washed after 30 minutes.

[0057] The result is as Figure 4 As shown, the results of flow cytometry show that miRNA-29a triggers the DNA walking reaction for 40 minutes, and ...

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Abstract

The invention discloses a multi-nucleic acid detection method and kit. The multi-nucleic acid detection method comprises the steps that DNA orbits and DNA walkers are designed, and the different DNA orbits are coupled to different fluorescent encoded microspheres; the DNA walkers are used as templates and the DNA orbits are used as primers for a DNA walking reaction, wherein target nucleic acid contained in a sample can trigger DNA walking on the surface of the fluorescent encoded microspheres; fluorescent labeling is performed on DNA walking products on the surface of the fluorescent encodedmicrospheres; and fluorescence detection of the fluorescent encoded microsphere is performed by using a flow cytometry to obtain the content of the target nucleic acid in the sample. According to themulti-nucleic acid detection method and kit, DNA walking is combined with the fluorescent encoded microspheres to realize rapid, highly sensitive and multivariate detection of the nucleic acid, and anew method is provided for detecting the nucleic acid such as miRNA.

Description

technical field [0001] The invention relates to nucleic acid detection, in particular to a multiplex nucleic acid detection method and kit. Background technique [0002] Lung cancer is a common cancer, and non-small cell lung cancer, which has a higher mortality rate, accounts for about 85% of lung cancer cases. Detection of tumor markers is considered to be a feasible method for early diagnosis of lung cancer. Among the tumor markers of non-small cell lung cancer, circulating tumor cells are inconsistent and require complex enrichment and isolation procedures; circulating DNA has false negatives; circulating miRNA has high stability and high reproducibility. Therefore, circulating miRNAs in blood are ideal biomarkers for non-invasive diagnosis of NSCLC. Current detection methods suffer from time-consuming and insufficient sensitivity. [0003] DNA molecules have the characteristics of self-assembly, programmability and predictability, and can build bionic DNA machines. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6834
CPCC12Q1/6834C12Q2563/149C12Q2563/131C12Q2563/107C12Q2525/301C12Q2525/207C12Q2521/101
Inventor 孙清江王琎屈晓君刘玉乾
Owner SOUTHEAST UNIV
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