Novel coronavirus Omicron mutation sequence detection technology based on multiple fluorescent quantitative ARMS-PCR technology and application thereof

A sequence and virus technology, which is applied in the field of detection of the new coronavirus Omicron nucleic acid mutation type, can solve the problems that are not related to the new coronavirus Omicron virus, etc., and achieve the effect of fast and objective detection results, good specificity, and pollution prevention

Inactive Publication Date: 2022-01-18
常州国药医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of the products currently on the market are ordinary new coronavirus detec

Method used

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  • Novel coronavirus Omicron mutation sequence detection technology based on multiple fluorescent quantitative ARMS-PCR technology and application thereof
  • Novel coronavirus Omicron mutation sequence detection technology based on multiple fluorescent quantitative ARMS-PCR technology and application thereof
  • Novel coronavirus Omicron mutation sequence detection technology based on multiple fluorescent quantitative ARMS-PCR technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 detection kit and its use

[0037] The kit includes the following components:

[0038] PCR reaction solution, enzyme mixture, mutant gene multiple reaction solution, internal standard reaction solution, positive control 1, negative control;

[0039] PCR reaction solution includes 10× buffer, 25mM MgCl 2 , 10mM dUTP and 10mM dNTPs; the enzyme mixture includes Taq enzyme, reverse transcriptase and UNG enzyme, the ratio of the upstream and downstream primers and probes of the mutant gene multiple reaction solution and the internal standard reaction solution is 4:4:1; The upstream primer is 300 nM, the downstream primer is 300 nM, and the probe is 100 nM.

[0040] See Table 1 for the nucleotide sequences of the primers and probes of the novel coronavirus mutant gene used in this example.

[0041] Table 1 Mutant gene multiplex reaction solution

[0042] 679NK-F TAGGGGCTGAATATGTCAACAACTC SEQ ID No.1 679NK-R CGTGCCCGCCGATGAGACT SEQ ID ...

Embodiment 2

[0099] The sensitivity test of embodiment 2 kit

[0100] The positive reference product is a non-replicating lentivirus of the corresponding mutant sequence of the new coronavirus S gene, which is synthesized by a professional organization; 10-fold gradient dilution to 500 copies per milliliter (the amplification curve jumping off on the right in the figure).

[0101] The kit of the present invention is used for detection.

[0102] The test results show that the kit of the present invention has good sensitivity for the detection of the corresponding mutation sequences of the S gene of the new coronavirus (446G>S, 547T>K, 496G>S, 679N>K), reaching 500 copies per milliliter, and CT The value changes in a gradient with decreasing concentration. See figure 1 , figure 2 , image 3 , Figure 4 .

Embodiment 3

[0103] The specificity test of embodiment 3 kits

[0104] In order to detect the specificity of nucleic acid detection kit (fluorescence PCR method) of the present invention, detect rhinovirus, enterovirus, adenovirus, respiratory syncytial virus, influenza virus (A and B type) with it (fluorescence PCR method) , parainfluenza virus, etc.

[0105] The test results show that the detection kit of the present invention can specifically amplify the corresponding mutant sequences (446G>S, 547T>K, 496G>S, 679N>K), without cross-reaction with nucleic acids of other viruses in the respiratory tract, see Figure 5 , Image 6 , Figure 7 , Figure 8 .

[0106] The above results show that the kit of the present invention is generally applicable clinically, has good sensitivity, high specificity, high accuracy rate, and saves time and labor in the operation process.

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Abstract

The invention provides a novel coronavirus Omicron mutation sequence detection technology based on a multiple fluorescent quantitative ARMS-PCR technology and application thereof. The novel coronavirus Omicron mutation sequence detection technology is mainly based on the fluorescent quantitative ARMS-PCR technology to perform single-tube or multi-tube multiple detection on the current Omicron mutant strain S gene specific mutation types, such as sequence position 23599, sequence change T exceeding G, sequence position 23048, sequence change G exceeding A, sequence position 23202, sequence change C exceeding A, sequence position 22898 and sequence change G exceeding A. A kit can well identify new key mutation of the currently popular novel coronavirus Omicron strain, has good specificity, and has very positive significance for novel coronavirus (Omicron) mutation monitoring.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the detection technology and application of the mutation type of the novel coronavirus Omicron nucleic acid based on multiple fluorescent quantitative ARMS-PCR technology. Background technique [0002] The new respiratory disease that broke out since December 2019 has more than 90 million confirmed cases worldwide as of mid-January 2021. The main clinical symptoms are fever, fatigue, and dry cough. After isolation and identification, the pathogen was confirmed to be a new type of coronavirus, which was named "SARS-CoV-2" by the International Committee on Taxonomy of Viruses. This is the seventh coronavirus currently found in the human body. There were mainly 6 types before, of which 229E, OC43, NL63, and HKU1 are less pathogenic and can cause mild respiratory diseases. The remaining two are SARS coronavirus and MERS. Coronavirus, which can cause severe respiratory illness. [0003]...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/16C12Q2600/166C12Q2535/137C12Q2537/143C12Q2563/107C12Q2545/113C12Q2521/107C12Q2521/531C12Q2545/101Y02A50/30
Inventor 孔祥宾李万帅汤丽丽
Owner 常州国药医学检验实验室有限公司
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