Screening method of SARS-CoV2 potential mutation sites and application thereof

A mutation site and screening method technology, applied in the fields of bioinformatics and biomedicine, can solve problems such as limited software functions, unsuitability for mutation analysis of large-scale data sets, and inability to realize virus mutation monitoring, etc., to achieve improved packaging efficiency and purity High and stable effect

Pending Publication Date: 2021-10-01
NANJING LEADING BIOMEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The software used in the above prediction methods has very limited functions, and they are not suitable for mutation analysis of large-scale data sets
Moreover, although the above prediction algorithm can output mutation sites in the nucleotide sequence, it cannot identify non-synonymous substitution sites that cause amino acid changes, and more importantly, its prediction method cannot realize the mutation monitoring of specific viruses

Method used

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  • Screening method of SARS-CoV2 potential mutation sites and application thereof
  • Screening method of SARS-CoV2 potential mutation sites and application thereof
  • Screening method of SARS-CoV2 potential mutation sites and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Screening method for potential mutation sites of SARS-CoV2

[0041] (1) For a specific new type of coronavirus SARS-CoV2, the whole genome sequencing data of the new crown is uploaded to the GISAID database (https: / / www.gisaid.org / ), and can be downloaded from the GISAID database for free. Use BioAide software to quickly annotate and compare the downloaded sequences, and extract the sequences of all coding genes from the whole genome sequence.

[0042] Sequence comparison: first drag the unaligned virus genome sequence directly to the input box, or load the file path through the file button;

[0043] Then select the sequence type as nucleotide (Nucleotide), the output format as fasta format, and select the alignment strategy as automatic mode (auto);

[0044] Finally, according to the configuration of the computer, select the number of threads to use, and then click the Run (Start) button to complete the comparison of the whole genome of the virus sequence se...

Embodiment 2

[0088] Example 2 Single point mutation example: S-S477N

[0089] Test materials: pcDNA3.1-SARS2-S-HA (diluted to 10 ng / µL), KOD high-fidelity enzyme KOD –Plus-Neo (Code: KOD-401), Dpni enzyme (NEB R0176S)

[0090] Primer design:

[0091] Table 4

[0092]

[0093] Design principles of point mutation primers:

[0094] 1. The length of the mutant primer is 35-40bp. Centering on the amino acid to be mutated, the forward and reverse primers have an overlapping region of 15-20bp.

[0095] 2. The GC content of the mutant primers is between 40-60%, especially at the 3' end.

[0096] The PCR amplification system is shown in Table 5:

[0097] table 5

[0098]

[0099] Mix and centrifuge gently.

[0100] The PCR amplification program is shown in Table 6:

[0101] Table 6

[0102]

[0103] PCR product identification:

[0104] Prepare 1% agarose gel and spot 5µL PCR product for observation. DNA marker 5000, the target band size is about 9kb. Result: the stripe size con...

Embodiment 3

[0112] Example 3 Example of multiple point mutation: SARS2-S-D614G+D1084Y

[0113] First make a single point mutant SARS2-S-D614G; then add the mutation site D1084Y on this basis.

[0114] Test materials: pcDNA3.1-SARS2-S-HA (diluted to 10 ng / µL), KOD high-fidelity enzyme KOD –Plus-Neo (Code: KOD-401), Dpni enzyme (NEB R0176S)

[0115] Primer design:

[0116] Table 8

[0117]

[0118] The PCR amplification system is shown in Table 9:

[0119] Table 9

[0120]

[0121] Mix and centrifuge gently.

[0122] The PCR amplification program is shown in Table 10:

[0123] Table 10

[0124]

[0125] PCR product identification:

[0126] Prepare 0.8% agarose gel and spot 5µL PCR product for observation. DNA marker 5000, the target band size is about 9kb. The result is as image 3 .

[0127] Dpni enzyme digestion: if there is a band in the identification result SARS2-S-D614G-1, add the remaining 15 µL of the PCR product to 0.4 µL of Dpni enzyme, mix well and place it at...

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Abstract

The invention relates to the technical field of bioinformatics and biological medicine, in particular to a screening method of SARS-CoV2 potential mutation sites. The method comprises the following steps of: 1) downloading to obtain a gene sequence of SARS-CoV2, carrying out rapid file annotation and sequence comparison on the downloaded sequence, and extracting sequences of all coding genes from a whole genome sequence; (2) calculating the mutation frequency of each site, screening out high-frequency mutation hot spots, and screening out mutation sites with remarkable selection advantages in a population by combining the sampling time and geographical distribution information of strains; 3) downloading tertiary structure information of proteins corresponding to existing coding genes; and 4) according to the predicted B cell and T cell epitopes, screening mutation sites on or near the immune epitopes, evaluating the possible influence of the mutation sites on the host immune response, and identifying the potential key mutation sites possibly related to virus infection and host adaptation of the SARS-CoV-2 on the genome in epidemic propagation.

Description

technical field [0001] The invention relates to the technical fields of bioinformatics and biomedicine, in particular a method for screening potential mutation sites of SARS-CoV2 and its application. Background technique [0002] In classification, coronaviruses belong to Nidovirales, Cornidovirineae, and Coronaviridae. They are enveloped, single-stranded, non-segmented RNA viruses. The coronavirus particle is spherical, with a diameter of about 120 nm and a genome of about 26-32 kb, which is the largest RNA virus in the known genome. Coronaviruses are phylogenetically classified into four genera: α-CoV, β-CoV, γ-CoV, and δ-CoV. The novel coronavirus (SARS-CoV-2) causes a new type of pneumonia, which often causes symptoms such as fever, cough, dyspnea, myalgia, and fatigue, and has had a huge impact on health and economy. The new coronavirus is mainly transmitted through saliva, droplets, contact, etc. At present, effective prevention and control is mainly carried out by c...

Claims

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Application Information

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IPC IPC(8): G16B20/30G16B20/50C12Q1/70G01N33/569G01N33/68C12N15/867C12N7/04C12N15/50C12R1/93
CPCG16B20/50G16B20/30C12Q1/701G01N33/56983G01N33/6878C12N15/86C12N7/00C07K14/005G01N33/6854C12Q2600/156G01N2333/165C12N2740/16043C12N2800/107C12N2770/20022C12N2770/20052C12N2770/20062G01N2469/20
Inventor 张玉基周秩建王琼王鹏张用科范文越徐达
Owner NANJING LEADING BIOMEDICAL TECH CO LTD
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