A kind of primer set, kit and application thereof for sars-cov-2 virus nucleic acid detection

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

Active Publication Date: 2021-11-16
HUBEI UNIV
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  • 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, especially the multi-channel fluorescent quantitative PCR instrument is expensive. It is about 250,000-300,000 yuan / unit. With the current detection throughput, each unit can only detect 3 batches per day, which seriously limits the detection throughput.

Method used

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  • A kind of primer set, kit and application thereof for sars-cov-2 virus nucleic acid detection
  • A kind of primer set, kit and application thereof for sars-cov-2 virus nucleic acid detection
  • A kind of primer set, kit and application thereof for sars-cov-2 virus nucleic acid detection

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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 the 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 Phosphorylation modification reaction system configuration 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 quencher 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 fluorophores and fluorescence quenching groups can also be used for labeling. The specific exc...

Embodiment 2

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

[0084] S1. Using the nucleic acid extract in the sample to be tested as a template, add separate N gene primers, ORF1ab gene primers, or add N gene primers and ORF1ab gene primers at the same time 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 is: 55°C, 15min; 85°C, 20min.

[0088]S2, using the above primer set, the cDNA containing a single target sequence (N gene or ORF1ab gene) obtained by the reverse transcription, and the cDNA containing both the N gene and the ORF1ab gene as templates, respectively performing single-plex PCR and double-PCR;

[0089] S3, mixing the guide DNA, molecular beacon and PfAgo e...

Embodiment 3

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

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

[0113] 2: Carry out PCR amplification to the positive standard substance of the different concentration obtained by step 1 dilution respectively, PCR amplification method is the same as embodiment 2;

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

[0115] The amplified products obtained by PCR were subjected to agarose gel electrophoresis, and the results were as follows: Figure 7 Shown; For the detection of fluorescence value of PfAgo digestion, such as Figure 8 shown. The results show that the fluorescence detection values ​​of eac...

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Abstract

The invention discloses a primer set, a kit and an application thereof for detection of SARS-CoV-2 virus nucleic acid. The present invention designs a primer set for the N gene and ORF1ab gene of the SARS-CoV-2 virus, and the kit includes the primer set, a guide DNA targeting the target gene, a fluorescently labeled molecular beacon and a PfAgo enzyme. The primer set provided by the invention can simply and quickly amplify the nucleic acid of the new coronavirus, 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 an application thereof for SARS-CoV-2 virus nucleic acid detection. Background technique [0002] At present, fluorescent quantitative PCR technology is the mainstream method for nucleic acid detection of new coronaviruses. The method is based on PCR technology. By designing a primer pair for the specific sequence of the novel coronavirus genome, the target nucleic acid fragment is specifically amplified. At the same time, a corresponding Taqman probe is added to the amplification system, and the two ends of the probe are Labeled with a fluorophore and a quencher, respectively. The amplification reaction is carried out in a fluorescent quantitative PCR instrument. As the thermal cycle proceeds, the probe bound to the single-stranded template will be cut by the exonuclease activity of DNA polymerase, and the fluorescent group will be se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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