Primer group and kit for nucleic acid detection of SARS-CoV-2 virus and application of primer group and kit

A sars-cov-2, primer set technology, applied in the field of biotechnology applications, can solve the problems of limited detection throughput and high price, and achieve the effects of good repeatability, strong specificity and high sensitivity

Active Publication Date: 2020-12-04
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the Taqman method has been popularized and provided a strong guarantee for the prevention and control of this epidemic, the nucleic acid detection method based on fluorescent PCR has high requirements for instruments and operators, esp

Method used

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  • Primer group and kit for nucleic acid detection of SARS-CoV-2 virus and application of primer group and kit
  • Primer group and kit for nucleic acid detection of SARS-CoV-2 virus and application of primer group and kit
  • Primer group and kit for nucleic acid detection of SARS-CoV-2 virus and application of primer group and kit

Examples

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preparation example Construction

[0070] 3. Preparation of guide DNA and molecular beacons

[0071] (1) Preparation of guide DNA:

[0072] The guide DNA guides the PfAgo enzyme to cleave the target DNA. All guide DNAs in the examples of the present invention were treated with T4 polynucleotide kinase (T4 PNK) to make the 5' end with a phosphate group. The T4 PNK reaction system is shown in Table 1.

[0073] Table 1 Configuration of phosphorylation modification reaction system of guide DNA

[0074]

[0075] (2) Preparation of molecular beacons:

[0076] In this embodiment, the 5' end and the 3' end of the molecular beacon are respectively modified by a fluorescent group and a quenching group, and the fluorescent group is selected from at least one of FAM, VIC, Cy5 or Rox; the The fluorescence quenching group is selected from at least one of BHQ-1, BHQ2, BHQ-3, BBQ or TAMRA. In other embodiments, all other known fluorescent groups and fluorescence quenching groups can also be used for labeling, and the sp...

Embodiment 2

[0083] The method for identifying or assisting in identifying whether a sample to be tested contains the novel coronavirus SARS-CoV-2 provided by the present invention includes the following steps:

[0084] S1. Using the nucleic acid extract in the sample to be tested as a template, separately add a single N gene primer, an ORF1ab gene primer, or add an N gene primer and an ORF1ab gene primer simultaneously for reverse transcription to obtain the cDNA of the new coronavirus SARS-CoV-2, The reverse transcription reaction system is shown in Table 4.

[0085] Table 4

[0086]

[0087] The reverse transcription reaction program was: 55°C, 15min; 85°C, 20min.

[0088]S2, adopt above-mentioned primer set, the cDNA that comprises single target sequence (N gene or ORF1ab gene) that described reverse transcription obtains and the cDNA that comprises N gene and ORF1ab gene simultaneously is template, carry out single-plex PCR and double PCR respectively;

[0089] S3, mixing the gui...

Embodiment 3

[0111] The present invention is used to identify or assist in identifying the detection limit of the detection method for the novel coronavirus SARS-CoV-2, comprising the following steps:

[0112] 1: Dilute the positive standard containing N gene to 1000copies / μL, 100copies / μL, 10copies / μL, 1copies / μL, 0copies / μL;

[0113] 2: PCR amplification was performed on the positive standards of different concentrations obtained by diluting in step 1, and the PCR amplification method was the same as that in Example 2;

[0114] 3: The PCR product amplified in step 2 is subjected to PfAgo digestion reaction, and the PfAgo digestion reaction is the same as that in Example 2;

[0115] Agarose gel electrophoresis was performed on the amplified products obtained by PCR, and the results were as follows: Figure 7 shown; for the detection of the fluorescence value of PfAgo digestion, such as Figure 8 shown. The results show that the fluorescence detection value of each dilution is significa...

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Abstract

The invention discloses a primer group and a kit for nucleic acid detection of SARS-CoV-2 virus and application of the primer group and the kit. The primer group for an N gene and an ORF1ab gene of the SARS-CoV-2 virus is designed, and the kit comprises the primer group, guide DNA targeting a target gene, a molecular beacon with a fluorescent marker and PfAgo enzyme. The primer group provided by the invention can be used for simply and quickly amplifying new coronavirus nucleic acid, and has the characteristics of good repeatability, high sensitivity and strong specificity.

Description

technical field [0001] The invention belongs to the application field of biotechnology, and in particular relates to a primer set, a kit and applications for the detection of SARS-CoV-2 virus nucleic acid. Background technique [0002] At present, fluorescence quantitative PCR technology is the mainstream method for the detection of new coronavirus nucleic acid. The method is based on PCR technology, and the target nucleic acid fragment is specifically amplified by designing primer pairs for the specific sequence of the novel coronavirus genome. At the same time, the corresponding Taqman probe is added to the amplification system, and both ends of the probe are Labeled with fluorophore and quencher group, respectively. The amplification reaction is carried out in a fluorescence quantitative PCR instrument. As the thermal cycle proceeds, the probe bound to the single-stranded template will be cleaved by the exonuclease activity of the DNA polymerase, and the fluorophore and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107
Inventor 马立新王飞何如怡翟超杨军刘洋余晓刘琳琳
Owner HUBEI UNIV
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