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Novel coronavirus fluorescence qRT-PCR method rapid detection system

A RT-PCR, coronavirus technology, applied in the field of rapid detection system of novel coronavirus fluorescence qRT-PCR method, can solve the problems of insufficient sensitivity and specificity, achieve the optimization of amplification system and amplification program, and ensure sensitivity , the effect of shortening the detection time

Active Publication Date: 2021-01-01
3D BIOMEDICINE SCI & TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, nucleic acid detection kits for detecting SARS-CoV-2 by qRT-PCR are already on the market, but the detection amplification time is not less than 60 minutes
The commercially available rapid nucleic acid detection kit (30min), the detection method is a constant temperature amplification method, and the sensitivity and specificity are not as good as the qRT-PCR detection method

Method used

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  • Novel coronavirus fluorescence qRT-PCR method rapid detection system
  • Novel coronavirus fluorescence qRT-PCR method rapid detection system
  • Novel coronavirus fluorescence qRT-PCR method rapid detection system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 Primer probe, reagent and detection method

[0063] 1.1 Primer probe sequence

[0064] The ORF1ab gene of the new coronavirus SARS-CoV-2 was selected as the target region for amplification, specific primers and fluorescent probes were designed, and the probes were labeled with FAM to detect whether the samples contained the new coronavirus RNA. Select the N gene of 2019-nCoV, design specific primers and fluorescent probes, and the probes are labeled with ROX to detect whether the samples contain 2019-nCoV RNA. Select the non-human genome and other species sequences that are not the detection target of the kit to design internal standard primer probes. The probes are labeled with VIC, and internal standard primer probes are added to the reaction system to monitor the entire experimental process.

[0065] 1.2 The reagents required for the detection of the new coronavirus SARS-CoV-2 are shown in Table 2, and the concentrations of each component are shown in T...

Embodiment 2

[0088] Embodiment 2 detection sensitivity

[0089] Use the primer-probe combination, reagent and nucleic acid amplification detection method of Example 1 to verify the sensitivity, detect the national standard product 2019-nCoV nucleic acid detection reagent national reference product (370099-202001), and the sensitivity reference product S concentration (stock solution) is 3 ×10 5 Copies / mL, jointly determined by digital PCR method. S using RNA / DNase-free deionized water for 1:3 dilution (2 parts water + 1 sample), 1:9, 1:27, 1:81, 1:243, 1:729, 1 :2187, 1:6561, 1:19683, 1:59049, and 1:177147 were marked as S1-S10 respectively, and were detected after nucleic acid extraction according to the kit instructions. According to the test results after serial dilution, the detection sensitivity of this kit to SARS-CoV-2 is 0.046copies / μL, such as figure 2 and image 3 shown.

Embodiment 3

[0090] Example 3 Negative coincidence rate (specificity)

[0091] The specificity was verified by using the primer-probe combination, reagents, and nucleic acid amplification detection method of Example 1.

[0092] In order to prove the cross-reactivity of the kit, the above reagents and nucleic acid amplification detection methods were used to verify the specificity of the detection system, and the national standard product 2019-nCoV nucleic acid detection reagent national reference (370099-202001)-negative reference product and No. National reference product (370007-201801) negative reference product of the second generation influenza A / B virus nucleic acid detection reagent.

[0093] Including the following pathogens: human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1, SARS coronavirus, MERS coronavirus, avian influenza virus H5N1, influenza B virus (BV), type A H1N1 (2009 ) influenza virus, influenza A H1N1 virus, influenza A H3...

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Abstract

The invention relates to a novel coronavirus fluorescence qRT-PCR method rapid detection system. The rapid detection method realizes optimization of an amplification system and an amplification program by optimizing a primer probe targeting ORF1ab genes and N genes of novel coronaviruses, enzyme mixed liquor (reverse transcriptase, DNA polymerase and UNG enzyme) of the amplification system, magnesium ion concentration and the like, so that the amplification time is shortened to be within 30 minutes. The detection time is greatly shortened, and meanwhile, the sensitivity, specificity, repeatability and accuracy of novel coronavirus detection are ensured.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a rapid detection system for novel coronavirus fluorescent qRT-PCR method. Background technique [0002] The 2019 novel coronavirus (SARS-CoV-2) belongs to the Betacoronavirus genus, and its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV. The pneumonia caused by SARS-CoV-2 infection is mainly transmitted by droplet and contact, and the population is generally susceptible. The general symptoms of SARS-CoV-2 infection are: fever, fatigue, dry cough, and gradually dyspnea. Some patients have mild onset symptoms, or even no obvious fever. Severe symptoms include: acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis, and coagulation dysfunction. In addition to the above symptoms, there may also be the following atypical symptoms: such as mild anorexia, fatigue, nausea and vomiting, dia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2521/107C12Q2563/107
Inventor 李丹董晋涛秦资李芳敏祝君巫益鸣陈才夫熊磊
Owner 3D BIOMEDICINE SCI & TECH CO LTD
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