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Primer group for rapid detection of 2019-nCoV based on CRISPR technology and application thereof

A 2019-ncov, detection primer technology, used in microorganism-based methods, recombinant DNA technology, and microbial assay/inspection. The effect of advantage

Active Publication Date: 2020-07-03
广州微远医疗器械有限公司 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in principle, the essence of its single-stage amplification reaction has not changed, and compared with most qPCR detection methods currently on the market, there is no substantial improvement in sensitivity

Method used

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  • Primer group for rapid detection of 2019-nCoV based on CRISPR technology and application thereof
  • Primer group for rapid detection of 2019-nCoV based on CRISPR technology and application thereof
  • Primer group for rapid detection of 2019-nCoV based on CRISPR technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Design of primer sequences for 2019-nCoVCRISPR detection.

[0057] 1. Target sequence selection.

[0058] Novel coronavirus (2019-nCoV) is a newly discovered coronavirus. The inventor analyzed the sequence of 2019-nCoV and believed that Orf1ab gene and N gene are conserved genes of coronavirus, which can be designed according to the conserved sequence, according to the target Spot the Orf1ab gene and N gene sequence regions, design crRNA and RPA amplification primers, and test the sensitivity of the detection method.

[0059] The sample to be tested in this embodiment is a plasmid (synthesized by Aiji Biotechnology Co., Ltd.) inserted with a selected target sequence region of 2019-nCoV. The sequence of Orf1ab gene insertion is as follows:

[0060] 5’-ATGCCTTCAAACTCAACATTAAATTGTTGGGTGTTGGTGGCAAACCTTGTATCAAAGT AGCCACTGTACAGTCTAAAATGTCAGATGTAAAGTGCACATCAGTAGTCTTACTCTCAGT TTTGCAACAACTCAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTACA CAATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTT...

Embodiment 2

[0072] 1. The amplification efficiency screening of RPA amplification primers.

[0073]In order to screen the RPA amplification primers of Cas13a, the plasmids inserted with the gene sequences of the two 2019-nCoV targets Orf1ab gene and N gene in the above-mentioned Example 1 were used as standard samples, and extracted according to conventional methods to obtain standard samples Genes, primers for the target Orf1ab gene (primers shown in the above table 1) are combined in two groups with Orf1ab-crRNA-1, and primers for the target N gene (primers shown in the above table 2) are paired Combined, and combined with N-crRNA-1 for detection and screening, the template concentration was 1000copies / μl.

[0074] 1.1 Method.

[0075] Reaction system: upstream primer (0.1-0.6μM) 0.5-2.5μL, downstream primer (0.1-0.6μM) 0.5-2.5μL, RPA enzyme master mix 21μL, magnesium acetate (10-20mM) 0.5-2μL, signal reporter probe (50-400nM) 1μl, NTP mixed solution (0.2-6mM) 1-10μL, T7 RNA polymeras...

Embodiment 3

[0097] In this example, sensitivity detection is performed based on RPA amplification, T7 in vitro transcription and Cas13a.

[0098] Using the plasmids with the conserved sequence of the nCoV-Orf1ab gene and the conserved sequence of the nCoV-N gene as templates, the calculated dilutions are 3000copies / μL, 300copies / μL, 30copies / μL, 3copies / μL, 1copy / μL, 0.4copy / μL , 0.16copy / μL, a total of 7 gradients were used as templates for sensitivity detection, and the amount of template added to each reaction was 2.5 μL.

[0099] 1. Method.

[0100] Referring to the method in Example 2 above, the sensitivity analysis was performed on the nCoV-Orf1ab target and the nCoV-N target respectively, and a negative control was set in the experiment.

[0101] Reaction conditions: react at 42°C for 40-60 minutes, and read the fluorescence value of FAM every 1 minute.

[0102] Refer to the result interpretation method in the above-mentioned embodiment 2 to perform result interpretation.

[010...

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Abstract

The invention relates to a primer group for rapid detection of 2019-nCoV based on a CRISPR technology and application thereof, and belongs to the technical field of gene detection of the CRISPR technology. The primer group comprises an Orf1ab gene amplification primer pair, an Orf1ab gene crRNA, an N gene amplification primer pair and an N gene crRNA. The primer group is adopted to detect 2019-nCoV by the CRISPR technology, the detection time of 2019-nCoV is shortened, and detection can be completed within 40-60min. A specific sequence combination is obtained through screening to serve as theprimer group for detection, the primer group has the advantages of being high in sensitivity and specificity, and the detection limit can reach 7.5copies. The primer group is adopted to conduct CRISPRdetection on 2019-nCoV, dependence on complex variable-temperature amplification instruments such as a qPCR instrument is eliminated, and the CRISPR-Cas technology has wide application prospects in the aspect of real-time diagnosis of 2019-nCoV.

Description

technical field [0001] The invention relates to the technical field of genetic detection based on CRISPR technology, in particular to a primer set for rapid detection of 2019-nCoV based on CRISPR technology and its application. Background technique [0002] Coronaviruses are a large class of RNA viruses. There are currently seven known coronaviruses that can infect humans. Four of these coronaviruses, including Human coronavirus 229E, Human coronavirus OC43, Human coronavirus NL63, and Human coronavirus HKU1, are more common in the population, but are less pathogenic and generally only cause mild respiratory syndrome similar to the common cold. Two other viruses, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome coronavirus (MERS), can cause severe respiratory illness. 2019-nCoV is the seventh human-infecting coronavirus discovered so far. [0003] 2019-nCoV is a betacoronavirus similar to MERS and SARS, both of which originated in bats. The in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/107C12Q2521/507C12Q2531/119C12Q2522/101
Inventor 许腾曾伟奇杨敏玲陈文景徐学中李永军王小锐苏杭
Owner 广州微远医疗器械有限公司
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