Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application of detection kit

A detection kit and coronavirus technology, which is applied in the field of clinical testing, can solve problems such as strengthening transmission, reducing vaccine effectiveness, and accelerating virus replication, achieving high sensitivity, improving detection accuracy, and shortening detection time.

Inactive Publication Date: 2021-08-06
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The L452R mutation will make the virus more transmissible and reduce the effectiveness of the vaccine
At the same time, some laboratory studies have found that the D614G mutation of the new coronavirus may lead to accelerated replicat...

Method used

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  • Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application of detection kit
  • Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application of detection kit
  • Detection kit for coronavirus SARS-CoV-2 and mutant strain thereof and application of detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the digital PCR detection kit of coronavirus SARS-CoV-2 and mutant strain thereof

[0042] The kit of the present invention comprises: digital PCR reaction solution, digital PCR primer mixture and negative control solution, combined with a fully automatic microdrop chip digital PCR system to complete the detection of coronavirus SARS-CoV-2 and its mutant strains.

[0043] in:

[0044] The components of the digital PCR reaction solution are 250mM PCR buffer, which is pH8.0, Tris-Hcl buffer, 20mM Mg 2+ Solution, 1 mM of each of the four dNTPs, 5 U of c-MMLV enzyme, 10 U of hot-start Taq enzyme, and 40 U of RNasin; the formula of the digital PCR reaction solution is shown in Table 1.

[0045] Table 1: Digital PCR Reaction Solution Recipe

[0046]

[0047]

[0048] The digital PCR primer mixture is the forward primer, reverse primer and primer used to detect the E484K, L452R, D614G sites in the coronavirus SARS-CoV-2 non-mutant strain ORF1ab gene, N ge...

Embodiment 2

[0067] The digital PCR detection kit for coronavirus SARS-CoV-2 and its mutant strains is used to detect the ORF1ab gene and N gene of the non-mutant strain of coronavirus SARS-CoV-2, and the S gene of the mutant strain of coronavirus SARS-CoV-2 The application in the biological samples of E484K, L452R, D614G sites, the methods involved in the detection of samples are:

[0068] (1) Nucleic acid sample extraction: take at least one of the subject's sputum, throat swab, and alveolar lavage fluid as a sample, use a commercially available RNA extraction kit, and follow the kit's instructions to extract RNA nucleic acid;

[0069] (2) The configuration of the PCR reaction system; the total volume of the PCR reaction system is 15 μL, the amount of digital PCR reaction solution added is 3 μL, and the amount of digital PCR primer mixture (SEQ ID NO: 1 to SEQ ID NO: 15) added is 2 μL, 3 μL for nucleic acid samples, and 7 μL for nuclease-free water;

[0070] (3) Generate droplets: trans...

Embodiment 3

[0079] Example 3: Detection of nucleic acid samples using a fully automatic micro-droplet chip digital PCR instrument produced by Shandong Laibo Biotechnology Co., Ltd.

[0080] (1) Nucleic acid sample extraction: Take at least one of the subject’s sputum, throat swab, and alveolar lavage fluid as a sample, use a commercially available RNA extraction kit (such as Qiagen’s QIAamp Viral RNA Mini Kit), and follow the reagent The instructions of the box are used to extract RNA nucleic acid;

[0081] (2) The configuration of the PCR reaction system; the total volume of the PCR reaction system is 15 μL, in which the amount of digital PCR reaction solution added is 3 μL, the amount of digital PCR primer mixture added is 2 μL, the amount of nucleic acid sample added is 3 μL, nuclease-free The amount of water added is 7 μL;

[0082] (3) Open the packing box in the ultra-clean workbench or biological safety cabinet, take out the droplet chip, and carefully tear off the outer package of...

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Abstract

The invention discloses a detection kit for coronavirus SARS-CoV-2 and a mutant strain thereof. The detection kit comprises a digital PCR reaction solution, a digital PCR primer mixed solution and a negative control solution, and is combined with a full-automatic microdroplet chip digital PCR system to detect the coronavirus SARS-CoV-2 and the mutant strain thereof. Specific primer probes are designed for two target genes of an ORF1ab gene and an N gene of a non-mutant strain of the coronavirus SARS-CoV-2 and three mutation sites of E484K, L452R and D614G in an S gene of the mutant strain of the coronavirus SARS-CoV-2, the detection accuracy of the kit is improved through digital PCR detection of different sites of multiple genes, and rapid judgment is achieved. The kit better meets clinical requirements in the detection, screening and vaccine development of the novel coronavirus, and also enables the detection to be faster and the application to be wider.

Description

technical field [0001] The invention relates to a virus detection kit and its application, in particular to a digital PCR detection kit for coronavirus SARS-CoV-2 and its mutant strain and its application, belonging to the technical field of clinical testing. Background technique [0002] The novel coronavirus, or "SARS-CoV-2", is a new strain of coronavirus that has never been found in humans before. Common signs after a person is infected with a coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. [0003] Closely tracking the mutant strains of the new coronavirus is now the focus of research by scientists around the world. The study of these mutations is conducive to the analysis of virus transmissibility and immune resistance. The new crown epidemic has lasted for a year. Judging from the current data, the mutati...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/166C12Q2537/143C12Q2563/159C12Q2563/107C12Q2545/113Y02A50/30
Inventor 陈振欧兰香朱成龙寇宗阳王岩贾秀玲苏真真高丽鹤
Owner 山东莱博生物科技有限公司
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