Primer, probe composition and kit for detecting novel coronavirus and variant thereof

A coronavirus and variant technology, applied in the field of bioengineering, can solve the problems of time-consuming gene sequencing, difficulty and large-scale promotion, and high cost.

Active Publication Date: 2021-12-21
成都峰际生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the mainstream method for detecting variant strains of the new coronavirus in the world is gene sequencing, but because gene sequencing is time-consuming and expensive, it is difficult to promote it on a large scale
Some manufacturers, such as Xiamen Zhishan Biotech and BGI Genomics, launched a new coronavirus mutation site detection kit based on the QPCR technology platform, which can only identify one mutant strain and cannot identify multiple different mutations at the same time strains, failed to meet the needs of clinical testing

Method used

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  • Primer, probe composition and kit for detecting novel coronavirus and variant thereof
  • Primer, probe composition and kit for detecting novel coronavirus and variant thereof
  • Primer, probe composition and kit for detecting novel coronavirus and variant thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This embodiment provides a variety of different primer / probe combinations for the detection of novel coronaviruses and their mutant strains

[0038] 1. Primer / Probe Set 1

[0039]

[0040] 1.1 The PCR reaction solution contains: 2×PCR reaction buffer, 0.1mmol / L~0.5mmol / L deoxyribonucleoside triphosphate, 0.2μmol / L~0.6μmol / L of the upstream and downstream primers of the target gene, and 0.2μmol / L~0.6μmol / L of the target gene Probe 0.1μmol / L~0.4μmol / L, internal standard gene upstream primer and downstream primer 0.1μmol / L~0.3μmol / L for amplification, internal standard probe 0.05μmol / L~0.2μmol / L.

[0041] 1.2 The PCR enzyme mixture contains: 1U / μl~5U / μl Taq DNA polymerase, 1U / μl~5U / μl reverse transcriptase, 1U / μl~10U / μl RNase inhibitor.

[0042] 1.3 This combination can detect the new coronavirus B.1.1.7 variant, B.1.351 variant or P.1 variant.

[0043] 2. Primer / Probe Combination II

[0044]

[0045] 2.1 The PCR reaction solution contains: 2×PCR reaction buffer, ...

Embodiment 2

[0059] This embodiment provides a method for using a detection kit for detecting novel coronavirus and its mutant strains

[0060] 1. Sample collection:

[0061] 1.1 Applicable samples: nasopharyngeal swabs, oropharyngeal swabs.

[0062] 1.2 Sample collection method: Use a swab to collect samples in the patient's nasopharynx or oropharynx, insert the swab to an appropriate depth, rotate it in situ for a few seconds to absorb the subject's secretions, and slowly rotate the swab out. Immediately after the sample is collected, put the swab into a virus sampling tube containing 1-3 ml of virus preservation solution.

[0063]1.3 The collected specimens should be sent for inspection in time, and those tested within 24 hours should be stored at 4°C.

[0064] 1.4 Samples should be transported using curlers with ice or foam boxes with ice for sealing.

[0065] 2. Sample pretreatment:

[0066] 2.1 Nucleic acid extraction from samples

[0067] Any commercial viral nucleic acid RNA e...

Embodiment 3

[0144] This embodiment provides the test of the novel coronavirus and its mutant strain detection reagent prepared by primer / probe combination one.

[0145] 1. Sample: The in vitro transcription product RNA1 of the DNA template (SEQ ID NO.33) contains the novel coronavirus ORF1 gene, N gene, S gene E484K mutation site, S gene 69 / 70Del mutation site, and S gene N501Y mutation site , the RNA fragment of the S253P mutation site of the ORF3A gene, the P71L mutation site of the E gene, and the in vitro transcription product RNA2 of the DNA template (SEQ ID NO.34) contains the RNA fragments of the ORF3A gene, the S gene, and the E gene.

[0146] 2. Method: According to the operation steps of Example 2, the method of the present invention dilutes the RNA fragments to a concentration of 3pg / ul, and performs parallel detection on different RNA fragments on the ABI 7500 instrument, and performs result analysis and statistics.

[0147] 3. Test results such as figure 1 shown.

[0148] 4...

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Abstract

The invention discloses a primer, a probe composition and a kit for detecting a novel coronavirus and a variant thereof. The primer and the probe composition comprise specific primers and probes aiming at an ORF1ab gene, an N gene and a reference gene of SARS-CoV-2, and further comprises specific primers and probes aiming at one or more mutation sites of 69-70Del, L452R, E484K, E484Q and N501Y mutation sites of an S gene of the SARS-CoV-2, 106/107/108del mutation sites of an NSP6 gene, P71L mutation sites of an E gene, V82A mutation sites of an ORF7A gene and S253P mutation sites of an ORF3A gene. The primer, the probe composition and the kit disclosed by the invention can be used for detecting mutation sites while detecting the novel coronavirus, and can be used for distinguishing one or more of a B.1.351 variant, a B.1. 1.7 variant, a B.1.617 variant or a P.1 variant of the novel coronavirus; and the kit is a novel coronavirus and variant detection reagent which is rapid, low in cost and rich in variation information, and provides powerful help for global epidemic situation prevention and control and epidemic disease research.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a primer, a probe composition and a kit for detecting novel coronaviruses and variant strains thereof. More specifically, it relates to a kit for nucleic acid detection of novel coronaviruses and mutant strains using multiplex fluorescent PCR technology. Background technique [0002] At present, the mainstream method for detecting new coronavirus mutant strains in the world is gene sequencing, but because gene sequencing is time-consuming and expensive, it is difficult to promote it on a large scale. Some manufacturers, such as Xiamen Zhishan Biotech and BGI Genomics, launched a new coronavirus mutation site detection kit based on the QPCR technology platform, which can only identify one mutant strain and cannot identify multiple different mutations at the same time strains, failed to meet the needs of clinical testing. Contents of the invention [0003] The technical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6858C12Q2600/156C12Q2600/166C12Q2600/16C12Q2531/113C12Q2563/107C12Q2537/143C12Q2545/101Y02A50/30
Inventor 刘亚宝韩英赵燕
Owner 成都峰际生物技术有限公司
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