COVID-19 and delta mutant strain detection kit and detection method thereof

A coronavirus and mutant technology, applied in the field of new coronavirus and its delta mutant detection kits, can solve the problems of prone to false negative results, poor accuracy, unfavorable prevention and treatment measures, etc.

Active Publication Date: 2021-11-02
北京吉检医疗科技有限公司
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  • Abstract
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Problems solved by technology

Therefore, when faced with samples that may contain the Delta strain, the existing novel coronavirus detection kits have limitations such as low detection sensitivity and poor accuracy, are prone to false negative result

Method used

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  • COVID-19 and delta mutant strain detection kit and detection method thereof
  • COVID-19 and delta mutant strain detection kit and detection method thereof
  • COVID-19 and delta mutant strain detection kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1 The preparation of each reagent

[0058] The purpose of the present invention is to screen the wild-type and delta mutant strains of the novel coronavirus, search for the full-length sequences of the ORF1ab and N genes of the novel coronavirus in GenBank, perform blast analysis on the ORF1ab and N genes separately, and compare them to obtain the target gene Conserved sequence segment, design the primer probe scheme in the conserved sequence segment of the gene, evaluate the Tm value, the difference between the Tm value of the target corresponding to the probe, GC content, avoid hairpin structure and dimer, etc. during the design process . It is used to distinguish the S gene of wild-type and mutant strains, search for the full-length sequence of the new coronavirus S gene in GenBank, perform blast analysis, select the L425R and T478K mutation sites, and try to avoid hairpin structures, primer internal dimers, The formation of dimers and mismatches between ...

Embodiment 2

[0066] Embodiment 2 Primer probe single verification

[0067] Take the wild-type pseudovirus, delta mutant pseudovirus and RP pseudovirus respectively, and use a commercial viral nucleic acid extraction kit to extract the nucleic acid to be tested as a template (4 concentration groups are set for each sample, corresponding to each target gene) PCR reaction system was prepared as follows: 15 μL of PCR reaction solution, 4 μL of primer-probe reaction solution (final concentration of upstream and downstream primers 50-500 nM, final concentration of probe 25-250 nM), Enzyme mixture 1 μL, template 5 μL, total volume 25 μL. Put the PCR tube into the fluorescence quantitative PCR instrument, and carry out the PCR reaction according to the following procedure: 50°C for 5 minutes; 95°C for 30s; 95°C for 5s, 66°C for 34s (collecting fluorescence), and cycle 45 reactions. The result is as Figure 1~Figure 4 and shown in Table 1.

[0068] Table 1 Amplification results of primer-probe s...

Embodiment 3

[0072] Embodiment 3 Mix fourfold

[0073] Four kinds of primer probes were combined into a quadruple system, and four different concentrations of delta mutant pseudoviruses were amplified, and the results were as follows: Figure 5 shown.

[0074] Table 2 Mixed quadruple validation amplification results

[0075]

[0076] *In order to simulate the real clinical sample collection process, according to past experience, the input concentration of fixed RP pseudovirus is 6×10 5 copies / mL.

[0077] This example illustrates that the quadruple system can detect different concentrations of delta mutant pseudoviruses, the amplification line is a standard S-shaped curve, and the Ct values ​​of the 10-fold diluted pseudoviruses are evenly spaced.

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Abstract

The invention provides COVID-19 and delta mutant strain detection kit and a detection method thereof, and belongs to the technical field of molecular biological detection. A series of primer probe groups are redesigned, and detection targets are increased, so that the novel coronavirus wild type and delta mutant strains are effectively distinguished. The kit can be used for in-vitro qualitative detection of COVID-19 or delta mutant strain infected pneumonia suspected cases and suspected aggregation case patients, and other patients needing COVID-19 infection diagnosis or identification of COVID-19 genes in nasopharyngeal swab, sputum and other samples of diagnosers.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a detection kit for a novel coronavirus and a delta mutant strain thereof and a detection method thereof. Background technique [0002] There have been many variants of the new coronavirus all over the world. Among them, the B.1.617 lineage is a variant virus first discovered in India in October 2020. On May 31, 2021, the World Health Organization named several major mutated virus strains of COVID-19 with Greek letters. The characteristic mutation sites of its sub-line B.1.617.2 are L452R and T478K, and it was named "Delta" variant. The Delta strain has a shorter incubation period (average 3.7 days), a higher viral load (1260 times higher than the original strain) required for the first nucleic acid test positive of an infected person, and a stronger Infectious (oropharyngeal swab infectious 80.65%) and other characteristics. Therefore, when fa...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 韩亚平张淼臧百盛顾城玮赵譞
Owner 北京吉检医疗科技有限公司
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