Primer and probe for detecting delta 69/70 HV deletion mutation site of S gene of new coronavirus Alpha strain and application of primer and probe

A technology of deletion mutation and virus, applied in the field of biomedicine, can solve the problems of high cost and long time consumption, and achieve the effect of high specificity and sensitive signal

Pending Publication Date: 2021-11-12
NANCHANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Whole-genome sequencing is the gold standard method for identif

Method used

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  • Primer and probe for detecting delta 69/70 HV deletion mutation site of S gene of new coronavirus Alpha strain and application of primer and probe
  • Primer and probe for detecting delta 69/70 HV deletion mutation site of S gene of new coronavirus Alpha strain and application of primer and probe

Examples

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Example Embodiment

[0028] Example 1. Primers and probes of new crown virus Alpha strain S gene A69 / 70HV missing mutant site

[0029] Due to the lack of amino acids 69 and 70 in the new coronavirus Alpha strain S gene, the ALPHA strain RT-QPCR method is carried out if the primer and probe designed based on the original strain s gene of the new crown virus may not be detected. New crown Alpha strain. The genome defective genotype is determined to be a reliable mark of an Alpha strain. The Alpha strain S gene Δ69 / 70HV lack sites designed primers and probes can be used as one of the effective proof of determining the Alpha strain.

[0030] Table 1. Primers used in the present invention, probe sequence

[0031] Sequence name Sequence information (5'-3 ') SEQ.ID.NO Alpha strain S gene forward primer CTTGGTTCCATGCTATCTCTCCTC 1 Alpha strain S gene reverse primer AttatgttagActorTcTcAgtggg 2 Original strain S gene forward primer Cttgtaatgtgtgaaggtg 3 Original strain S gene...

Example Embodiment

[0033] Example 2. Novel Coronary Virus Alpha strain S gene Δ69 / 70HV deletion mutant site detection

[0034] First, detect the required reagent

[0035] 1) Reagent I (1 ml / tube): Fluorescence quantitative PCR reaction buffer.

[0036] 2) Reagent II (lyophilized): At the same time, an Alpha strain and a primitive strain primer were added, the mixture of the probe (the primer concentration was 0.2 μmol / L, the concentration of the probe was 0.2 μmol / L).

[0037] 3) Reagents III: Premix EX TAQTM enzyme mixture (TAKARA).

[0038] 4) Yang - nature controls: The synthesis of new coronavirus Alpha strain S gene purpose fragments and fragments including new coronavirin primer strains.

[0039] 5) Yin properties control: water treated with diethyl pyrophosphate.

[0040] 6) Specimen type: nasal swab, throat swab, alveolar lavage.

[0041] Second, nucleic acid extraction

[0042]The commercial RNA extraction kit is used, such as a silica gel film centrifugal method or a magnetic bead ...

Example Embodiment

[0055] Example 3. Specific detection of primers and probes

[0056] The RNA fragment containing new crown virus Alpha strain, new crown virus raw toxic strain, SARS, and MERS coronavirus, respectively, according to the steps in Example 2, respectively. figure 1 with figure 2 As shown, the results show that the new crown virus Alpha strain is added, and the reaction tube of the new crown virus raw strain has occurred in FAM and HEX fluorescence paths, respectively. In the reaction tube of the RNA fragment of SARS and MERS coronavirus, the RNA fragment of the MERS coronavirus, whether it is FAM, or the HEX channel CT value is greater than 30 (displayed is negative), indicating the design of new crown virus Alpha strain, new crown virus raw toxicity Plant primers and fluorescent probes have high reaction specificity.

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Abstract

The invention discloses a primer and a probe for detecting a delta 69/70HV deletion mutation site of an S gene of a new coronavirus Alpha strain and application of the primer and the probe, and belongs to the technical field of biology. The primer sequence and the fluorescent probe sequence of the delta 69/70HV deletion mutation site on the S gene in the Alpha mutant strain are optimized, the optimized sequence has the advantages of high specificity, signal sensitivity and the like, whether the new coronavirus strain to be detected has the delta 69/70HV deletion S gene or not can be effectively and rapidly detected through the delta 69/70del primer and the probe, and the method can be used as one of effective evidences for judging whether a new coronavirus strain to be detected is an Alpha strain or not, and provides a detection means for judging whether the new coronavirus strain to be detected is an Alpha mutant strain or not. After a mixture of S gene amplification primers and fluorescent probes of the new coronavirus Alpha strain and the original strain is simultaneously added into a reaction tube, PCR detection can be carried out respectively without arranging two reaction tubes, and whether a sample to be detected is the S gene of the original strain or the S gene delta 69/70HV deletion type of the Alpha strain can be identified in the same reaction tube.

Description

technical field [0001] The invention belongs to the field of biomedical technology, and in particular relates to primers, probes and applications thereof for detecting the Δ69 / 70HV deletion mutation site of the S gene of a novel coronavirus Alpha strain. Background technique [0002] On December 14, 2020, the World Health Organization (WHO) reported a more contagious mutant strain of the new coronavirus, and named it B.1.1.7. The mutant strain is newly named Alpha mutant strain. Due to its huge transmission advantage, the Alpha mutant strain quickly spreads faster than the original strain of the new coronavirus. According to reports, compared with the new coronavirus strain (NCBI: NC_045512), the Alpha mutant strain has 23 mutations, including 14 non-synonymous mutations (changes in amino acids) and 6 synonymous mutations (no changes in amino acids). and 3 deletion mutations, respectively located in ORF1ab, N gene, ORF8 gene and S (Spike) gene, two of the 3 deletion mutati...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 何庆华
Owner NANCHANG UNIV
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