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Detection method of target nucleic acid and application thereof

A detection method and target nucleic acid technology, applied in the field of target nucleic acid detection, can solve problems such as prolonging detection time

Pending Publication Date: 2022-03-08
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This virus detection technology requires nucleic acid signal amplification upstream of the target sequence. Although this signal amplification process improves the sensitivity of detection, it usually requires early nucleic acid amplification technology to ensure that the reaction result is sufficiently obvious, which prolongs the time. detection time, therefore, it is necessary to develop a detection technique capable of both high accuracy and sensitivity

Method used

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  • Detection method of target nucleic acid and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the preparation of albumen

[0045] This embodiment relates to 3 kinds of plasmids of the III-A type CRISPR system, as follows:

[0046] The first method: using the pCDF plasmid vector as a template, five subunit proteins (including TtCsm1, TtCsm2, TtCsm3, TtCsm4, and TtCsm5) in the TtCsm complex are fused and expressed, and site-directed mutation is carried out, the 34th amino acid of Csm3 is asparagus Amino acid D is mutated to alanine A (D34A) to inactivate target RNA cleavage mediated by Csm3.

[0047] The second: using pACYC as a plasmid vector, inserting TtCsm1 (Cas10) protein and an artificially designed CRISPR array, the synthetic CRISPR array can recognize the N gene of SARS-CoV-2.

[0048] The third method: use pC0075 as a plasmid vector, insert the TtCsm6 protein sequence and fuse the SUMO tag to facilitate purification.

[0049] Co-transform Escherichia coli BL21(DE3) with the first plasmid and the second plasmid, grow in LB broth at 37°C to ...

Embodiment 2

[0051] Example 2: Preparation of fluorescent chip

[0052] This embodiment provides a glass slide anchored with a fluorescent reporter group, and the specific steps are as follows:

[0053] (1) Cover the surface of coverslip (24×30mm) with biotin through chemical modification: first, wash the slide, etch with acetone for 30min, then corrode with potassium hydroxide for 1h, rinse with distilled water Then dry under nitrogen; soak in aminosilane solution for 10 minutes in a dark environment, sonicate for 1-2 minutes and then return to a dark environment for 10 minutes; add biotin to the surface of the slide in bicarbonate buffer and incubate overnight ; Store at -80°C.

[0054] (2) Add streptavidin (streptavidin) to combine with biotin: use tape cut into thin strips on the surface of the slide to form a loading area, add streptavidin to the surface of the slide and incubate for 10 min, Residual streptavidin not bound to biotin was rinsed with distilled water.

[0055] (3) Anc...

Embodiment 3

[0056] Embodiment 3: the detection of target nucleic acid

[0057] This embodiment provides a method for detecting target nucleic acids for non-disease diagnosis purposes, comprising the following steps:

[0058] (1) Before the TtCsm complex, TtCsm6 protein and other reaction systems in Example 1 are added to the fluorescent chip slides prepared in Example 2, first use TIRFM to perform fluorescence excitation and imaging on the slides, and record the initial fluorescence intensity imaging.

[0059] (2) Add 250μM ATP, 500nM TtCsm complex, 2500nM TtCsm6 protein, target nucleic acid sample and reaction buffer (20mM Tris-HCl, pH 7.9, 200mM Monopotassium Glutamate, 10mM Ammonium Sulfate, 5mM Magnesium Sulfate , 1mM TCEP) and incubated for 5min, washed the sheared fluorescent reporter gene with distilled water and imaged again with TIRFM to obtain the fluorescence intensity after the reaction. concentration of target nucleic acid.

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Abstract

The invention discloses a target nucleic acid detection method and application thereof. In the first aspect, the invention provides a method for detecting target nucleic acid with a non-disease diagnosis purpose, and the method comprises the following steps: providing a substrate on which an RNA chain marked with a fluorescent reporter group is fixed; carrying out contact reaction on the Csm complex, Csm6 protein, a nucleic acid sample and a substrate, and washing the substrate after the reaction is finished; and respectively collecting fluorescence signals of the substrate before and after the reaction by using a total internal reflection fluorescence microscope, and obtaining the concentration of the target nucleic acid in the nucleic acid sample according to the fluorescence intensity change. The Csm complex recognizes the target nucleic acid to generate cA4, and further activates the activity of the non-specific cleavage single-stranded RNA of the Csm6 protein, so that the RNA chain anchored on the substrate is cleaved, the fluorescent reporter gene is dissociated along with the cleavage, and the amount of the fluorescent reporter group on the substrate is reduced. The high signal-to-noise ratio of the TIRFM is utilized to obtain obvious signals, and the accuracy and sensitivity of detection are improved.

Description

technical field [0001] The present application relates to the technical field of nucleic acid detection, in particular to the detection method and application of target nucleic acid. Background technique [0002] Through rapid and efficient detection, the new coronavirus SARS-CoV-2 and its variants can be detected in mild and asymptomatic infected persons in time, which can facilitate early diagnosis, treatment and follow-up of close contacts, and expand accurate and effective diagnosis scope of test. Therefore, the development and improvement of rapid, accurate, low-cost and scalable detection technologies for the novel coronavirus SARS-CoV-2 and its variants (i.e., RNA viruses) are particularly important for the growing public health needs. Most of the RNA detection of the early novel coronavirus SARS-CoV-2 relied on reverse transcription-polymerase chain reaction (RT-PCR), but this method is slow and requires complex and cumbersome instrument support. In contrast, the l...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/682C12Q1/6834C12R1/93
CPCC12Q1/70C12Q1/682C12Q1/6834C12Q2521/327C12Q2563/107C12Q2565/601
Inventor 刘畅悦秦培武何倩袁曦陈群
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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