Unlock instant, AI-driven research and patent intelligence for your innovation.

A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system

A coronavirus and detection method technology, applied in the field of novel coronavirus dual-target rapid detection method and kit, to achieve the effect of overcoming incompatibility

Active Publication Date: 2022-04-12
ANHUI MEDICAL UNIV
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DETECTR and SHERLOCK systems based on the CRISPR / Cas system can only detect a single target at present.
However, the experiment found that each different Cas protein can only cut the corresponding single-stranded DNA probe (LbaCas12a) or single-stranded RNA probe (LwaCas13a) after being activated, which makes the integration of the two CRISPR systems into a single system for dual-target detection. Simultaneous detection possible

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system
  • A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system
  • A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: A novel coronavirus dual-target rapid detection method and kit based on CRISPR / Cas system

[0057] Kit includes:

[0058] Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

[0059] Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

[0060] Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

[0061] Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

[0062] Cas12a-N-crRNA:

[0063] 5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

[0064] Cas13a-S-crRNA:

[0065] 5'-gauuuagacuaccccaaaaacgaagggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACACC;

[0066] RNA fluorescent probe: 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';

[0067] Wherein, m is 2-position oxygen methyl modification, r is RNA;

[0068] DNA fluorescent probe: 5'-VIC-TTATTATT-BHQ1-3';

[0069] buffer A solution, BufferB solution, HEPES buffer, MgCl 2 Solution, 10×NEB buffer2.1 buffer, RNase inhibitor, T7 RNA polymerase and RNase-free water.

[0070] The preferred techn...

Embodiment 2

[0147] Example 2: A novel coronavirus dual-target rapid detection method and kit based on CRISPR / Cas system

[0148] A kit for rapid detection of novel coronavirus double targets based on CRISPR / Cas system, said kit comprising:

[0149] Two sets of RT-RAA amplification primers:

[0150] Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

[0151] Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

[0152] Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

[0153] Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

[0154] Two crRNAs specifically detected by CRISPR:

[0155] Cas12a-N-crRNA:

[0156] 5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

[0157] Cas13a-S-crRNA:

[0158] 5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACCC-3';

[0159] RNA fluorescent probe: 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';

[0160] Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

[0161]...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

A novel coronavirus dual-target rapid detection method and kit based on the CRISPR / Cas system, including nucleic acid extraction to obtain the nucleic acid to be detected; two sets of RT-RAA primers are used to simultaneously amplify two sites to be detected and obtain nucleic acid products, respectively It is the amplification product of the N gene targeted by Cas12a and the amplification product of the S gene targeted by Cas13a; prepare a CRISPR reaction mixture, add 2 μL of the nucleic acid amplification product to the CRISPR reaction mixture, incubate at 37°C for 30 minutes, and obtain by reading the fluorescence Test results. The invention has the advantages of fast detection speed, high accuracy and low cost. The present invention overcomes the incompatibility between the DETECTR and SHERLOCK systems, and successfully unifies the LwaCas13a derived from Cytomonas varvernerii and the LbaCas12a derived from Streptomyces ND2006 without reducing the sensitivity and accuracy of the detection method, realizing In order to use the CRISPR system to detect two target sites simultaneously.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a novel coronavirus dual-target rapid detection method and a kit based on a CRISPR / Cas system. Background technique [0002] Novel coronavirus (COVID-19), referred to as "new coronavirus", is a newly discovered single-stranded RNA virus with a total length of 29,903 nucleotides. It is transmitted through the respiratory tract and conjunctiva through droplets. It is highly infectious and spreads widely. , is currently the seventh coronavirus known to be pathogenic to humans. Compared with the acute symptoms caused by other coronaviruses, the symptoms of 2019-nCoV infection range from mild, cough, fever to critical illness. The symptoms of infection are similar to common respiratory diseases, and they are highly concealed and highly contagious. [0003] At present, there is no effective therapeutic drug for the new crown virus. The development of the new crown vaccine r...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/107C12Q2521/507C12Q2521/327C12Q2525/161C12Q2563/107
Inventor 杜忆南邢晨柳燕周静祝亚亭王小凤李倩张智康
Owner ANHUI MEDICAL UNIV