Novel coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application of kit

A virus nucleic acid and kit technology, applied in the field of new coronavirus nucleic acid and mutant gene detection kits, can solve problems such as the inability to detect ordinary new coronavirus mutant strains

Pending Publication Date: 2021-05-18
常州国药医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the products currently on the market are ordinary new coronavirus detection products. One PCR reaction tube can only detect ordinary new coronavirus in one reaction, and cannot specifically detect common new coronavirus and highly contagious new coronavirus mutant strains at the same time.

Method used

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  • Novel coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application of kit
  • Novel coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application of kit
  • Novel coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 detection kit and its use

[0044] The kit includes the following components:

[0045] PCR reaction solution, enzyme mixture, ORF1ab / N / internal reference / S mutant gene multiple reaction solution, positive control 1, negative control;

[0046] The PCR reaction solution includes 10× buffer, 25mM MgCl2, 10mM dUTP and 10mM dNTPs; the enzyme mixture includes Taq enzyme, reverse transcriptase and UNG enzyme, ORF1ab / N / internal reference / S mutant gene multiple reaction solution upstream and downstream primers, and The ratio of the probes is: 4:4:1; the preferred upstream primer is 400nM, the downstream primer is 400nM, and the probe is 100nM.

[0047] The nucleotide sequences of the primers and probes of the novel coronavirus ORF1ab / N / internal reference / S mutant gene used in this example can be found in Table 1.

[0048] Table 1 Nucleotide sequence list of primers and probes of the new coronavirus ORF1ab / N / internal reference / S mutant gene in the present inventi...

Embodiment 2

[0106] Embodiment 2 detection kit and its use

[0107] The kit includes the following components:

[0108] PCR reaction solution, enzyme mixture, ORF1ab / N / internal reference gene multiple reaction solution, S mutation gene reaction solution, positive control 1, positive control 2, negative control;

[0109] The PCR reaction solution includes 10× buffer, 25mM MgCl2, 10mM dUTP and 10mM dNTPs;

[0110] Enzyme mix includes Taq enzyme, UNG enzyme, reverse transcriptase

[0111] The ratio of upstream and downstream primers and probes of ORF1ab / N / internal reference gene multiplex reaction solution is: 4:4:1; the preferred upstream primer is 400nM, the downstream primer is 400nM, and the probe is 100nM.

[0112] The ratio of upstream, downstream primers and probes in the S mutation gene reaction solution is: 4:4:2; the preferred upstream primer is 400nM, the downstream primer is 400nM, and the probe is 200nM.

[0113] The positive control includes 2 combinations, the components a...

Embodiment 3

[0149] The sensitivity test of embodiment 3 kit

[0150] Positive control reference products are the non-replicating lentivirus of the A23063T mutant sequence of the ORF1ab, N, and S genes of the new coronavirus and the non-replicating adenovirus of part of the human β-actin gene sequence, synthesized by professional institutions; 10-fold gradient dilution to 30 copies per milliliter ( figure 1 Amplification curve with jump in middle right).

[0151] The negative reference product is RNase-free water, 1000ml of RNase-free water, then sterilized in a sterilizing pot at 121°C for 20 minutes, marked and stored at room temperature.

[0152] The kit of the present invention is used for detection. The detection method is the same as in Example 1.

[0153] The test results show that the kit of the present invention has good sensitivity for the detection of the A23063T mutation sequence of ORF1ab, N, and S genes of the new coronavirus, reaching 30 copies per milliliter, and the CT ...

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Abstract

The invention provides a novel coronavirus nucleic acid and mutant gene detection kit based on a fluorescent quantitative PCR technology and application of the kit. The kit is mainly used for detecting the S gene mutation type N501Y on the basis of the fluorescent quantitative PCR technology. A target gene can be detected by using primers as shown in SEQ ID No.10 and SEQ ID No.11 and a probe with a base sequence as shown in SEQ ID No.12. The sensitivity reaches 30 copies per milliliter, the specificity is good, and the kit has very positive significance on the detection of novel coronavirus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and its application. Background technique [0002] The new respiratory disease that broke out since December 2019 has more than 90 million confirmed cases worldwide as of mid-January 2021. The main clinical symptoms are fever, fatigue, and dry cough. After isolation and identification, the pathogen was confirmed to be a new type of coronavirus, which was named "SARS-CoV-2" by the International Committee on Taxonomy of Viruses. This is the seventh coronavirus currently found in the human body. There were mainly 6 types before, of which 229E, OC43, NL63, and HKU1 are less pathogenic and can cause mild respiratory diseases. The remaining two are SARS coronavirus and MERS. Coronavirus, which can cause severe respiratory illness. [0003] The genome size of the new coronavirus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/114Y02A50/30
Inventor 李万帅孔祥宾湯丽丽
Owner 常州国药医学检验实验室有限公司
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