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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

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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whole-genome sequencing is the gold standard method for identifying virus strains, however, it is time-consuming and expensive

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

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Primers and probes for the S gene A69 / 70HV deletion mutation site of the new coronavirus Alpha strain

[0029] Due to the deletion of amino acids 69 and 70 in the S gene of the new coronavirus Alpha strain, if primers and probes designed based on the S gene of the original new coronavirus strain are used to detect the Alpha strain by RT-qPCR, it may not be detected New crown Alpha strain. After whole-genome sequencing verification, the S-gene target failure genotype (S-gene target failure, SGTF) was identified as a reliable marker for the Alpha strain. Select the S gene Δ69 / 70HV deletion site sequence of the Alpha strain to design primers and probes, which can be used as one of the effective proofs for determining the Alpha strain.

[0030] Table 1. Primers and probe sequences used in the present invention

[0031] sequence name Sequence information (5'-3') SEQ.ID.No Alpha strain S gene forward primer CTTGGTTCCATGCTATCTC 1 Alpha s...

Embodiment 2

[0033] Example 2. Detection of the S gene Δ69 / 70HV deletion mutation site of the new coronavirus Alpha strain

[0034] 1. Reagents required for detection

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

[0036] 2) Reagent II (lyophilized): a mixture of primers and probes for both the Alpha strain and the original strain (the concentration of the primer pair is 0.4 μmol / L, and the concentration of the probe is 0.2 μmol / L).

[0037] 3) Reagent III: Premix EX TaqTM enzyme mixture (TAKARA company).

[0038] 4) Positive quality control: including the synthetic new coronavirus Alpha strain S gene target fragment and including the new coronavirus original strain S gene target fragment.

[0039] 5) Negative quality control: water treated with diethyl pyrophosphate.

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

[0041] 2. Nucleic acid extraction

[0042]Use a commercial RNA extraction kit, such as a silica membrane spi...

Embodiment 3

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

[0056] Add RNA fragments containing the full-length S gene of the new coronavirus Alpha strain, the original new coronavirus strain, SARS and MERS coronavirus respectively to the reaction tube, and detect according to the steps in Example 2, such as figure 1 and figure 2 As shown, the results show that amplification curve signals appeared in the FAM and HEX fluorescence channels respectively in the reaction tubes added with the Alpha strain of the new coronavirus and the original strain of the new coronavirus. In the reaction tubes added with RNA fragments of the full-length S gene of SARS and MERS coronaviruses, the Ct values ​​of both FAM and HEX channels were greater than 30 (shown as negative), indicating that the designed Alpha strain of the new coronavirus, the original virus of the new coronavirus The strain 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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