Method for detecting novel coronavirus variants and subtypes

A technology for coronaviruses and mutant strains, applied in the fields of probes, detection systems, and specific crRNA, can solve the problems of high technical and professional knowledge requirements for operators, high configuration requirements, and long time-consuming, etc., to achieve shortened detection time, high sensitivity, The effect of easy operation

Active Publication Date: 2021-08-06
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, virus gene sequencing requires high laboratory configuration, time-consuming, high cost, and extremely high requirements for operator skills and expertise, which limits its application in the rapid identification of SARS-CoV-2 virus variants

Method used

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  • Method for detecting novel coronavirus variants and subtypes
  • Method for detecting novel coronavirus variants and subtypes
  • Method for detecting novel coronavirus variants and subtypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] A specific crRNA used to identify the nucleic acid of different coronavirus SARS-CoV-2 variants, based on CRISPR-Cas12a technology detection,

[0067] The specific crRNA used to identify the L subtype and the S subtype is Orf8-crRNA, and the nucleotide sequence is 5'-UAAUUUCUACUAAGUGUAGACCUUUUACAAUUAAUUGCCA-3' (SEQ ID NO 1).

[0068] The specific crRNAs used to identify the mutation sites L5F, D80A, D215G, R246I, Y453F, N501Y, A570D, D614G, A701V, T716I, S982A, P1263L, K417N, L452R, and E484Q of the S protein gene of SARS-CoV-2 are S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA, S-R246I-crRNA, S-Y453F-crRNA, S-N501Y-crRNA, S-A570D-crRNA, S-D614G-crRNA, S- A701V-crRNA, S-T716I-crRNA, S-S982A-crRNA, S-P1263L-crRNA, S-K417N-crRNA, S-L452R-crRNA, S-E484Q-crRNA, the nucleotide sequences are respectively:

[0069] S-L5F-crRNA: 5'-UAAUUUUCUACUAAGUGUAGAUUUUAUUGCCACUAGUCUCU-3' (SEQ ID NO2);

[0070] S-D80A-crRNA: 5'-UAAUUUCUACUAAGUGUAGACUAACCCUGUCCUACCAUUU-3' (SEQ ID NO.3);

[0071] ...

Embodiment 2

[0107] A CRISPR-Cas12a detection system for detecting variants of SARS-CoV-2, including the specific crRNA in Example 1, probes, nuclease-free water and buffer buffer2.1.

[0108] The detection system is detected through the following steps:

[0109](1) Using the detection system, the detection system includes 0.4 μL of crRNA with a concentration of 15 μM, 1 μL of Cas12a with a concentration of 10 μM, 1 μL of a reporter probe with a concentration of 10 μM, 1× buffer buffer 2.1 27.6 μL, and placed at a constant temperature of 37°C After incubation in the fluorescence detector for 10 minutes, add SARS-CoV-2 genomic DNA as a template for CRISPR-Cas12a detection, the detection time is 30 minutes, and the detection result is obtained;

[0110] (2) Analyze the fluorescent signal to determine whether the sample contains SARS-CoV-2 variant virus nucleic acid. Fluorescent signals are detected and judged as positive, indicating that there is nucleic acid of the corresponding variant st...

Embodiment 3

[0117] The CRISPR-Cas12a detection method of the present invention is used to identify SARS-CoV-2-Orf8-L type / S type.

[0118] This example is used to detect the CRISPR-Cas12a detection system of SARS-CoV-2 mutant strains, which contains SARS-CoV-2 specific crRNA, universal probe, nuclease-free water, and buffer 2.1. Buffer buffer 2.1 (New England BioLabs, Ipswich, M0653T), nuclease-free water (Aikerui Bio, AG11012). crRNA (synthesized by Qingke Biotechnology Co., Ltd.) was used at a concentration of 15 μM, Cas12a (New England BioLabs, Ipswich, M0653T) was used at a concentration of 10 μM, and the probe (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.) was used at a concentration of 10 μM. 1× buffer buffer2.1.

[0119] crRNA and probe sequences are as follows:

[0120] Orf8-crRNA:5'-UAAUUUCUACUAAGUGUAGACCUUUUACAAUUAAUUGCCA-3'(SEQ ID NO.1)

[0121] Probe sequence: 5'-TTATT-3' (SEQ ID NO.14)

[0122] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize ...

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Abstract

The invention relates to a specific crRNA, probe, detection system and method for detecting a novel coronavirus SARS-CoV-2 variant based on CRISPR-Cas12a method. The specific crRNA for identifying an L subtype and an S subtype is Orf8-crRNA, and the specific crRNA for identifying variation sites of L5F, D80A, D215G, R246I, Y453F, N501Y, A570D, D614G, A701V, T716I, S982A, P1263L, K417N, L452R and E484Q of SARS-CoV-2 S protein genes is S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA, S-R246I-crRNA, S-Y453F-crRNA, S-N501Y-crRNA, S-A570D-crRNA, S-D614G-crRNA, S-A701V-crRNA, S-T716I-crRNA, S-S982A-crRNA, S-P1263L-crRNA, S-K417N-crRNA, S-L452R-crRNA and S-E484Q-crRNA. The nucleotide sequence of the probe is SEQ ID NO. 14. The detection system comprises crRNA, the probe, ribozyme-free water and a buffer solution buffer 2.1. The specific crRNA, the probe, the detection system and the method can accurately detect the SARS-CoV-2 variants, do not need to depend on expensive instruments and professional technicians, are short in detection time, and have the advantages of high specificity, high sensitivity, rapidness, high efficiency, simplicity and convenience in operation, readily readable results and the like.

Description

technical field [0001] The invention relates to the technical field of in vitro virus detection, in particular to a specific crRNA, a probe, a detection system and a method for detecting novel coronavirus SARS-CoV-2 mutant strains based on the CRISPR-Cas12a method. Background technique [0002] At present, the new coronavirus SARS-CoV-2 has evolved into a variety of mutant strains and subtypes, resulting in changes in virus pathogenicity and transmission efficiency, affecting the protection rate of vaccines, and becoming a new problem in epidemic prevention and control. Although the variant strains and subtypes of SARS-CoV-2 can be discovered and diagnosed through virus genome sequence analysis, it cannot meet the needs of on-site detection, nor can it achieve high-throughput detection of a large number of samples, which affects epidemic prevention and control. [0003] The two types of the new coronavirus SARS-CoV-2 are the L subtype and the S subtype. The difference betwee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/70C12Q1/6816C12R1/93
CPCC12N15/1131C12Q1/701C12Q1/6816C12N2310/20C12Q2521/327C12Q2525/161C12Q2525/151C12Q2563/107Y02A50/30
Inventor 唐时幸梁源浩赵建辉王海鹰
Owner SOUTHERN MEDICAL UNIVERSITY
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