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Novel coronavirus detection kit and detection method thereof

A technology for virus detection and kits, applied in biochemical equipment and methods, measurement/testing of microorganisms, resistance to vector-borne diseases, etc., can solve problems such as sensitivity and accuracy need to be improved

Pending Publication Date: 2020-12-08
北京吉检医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This all reflects that the sensitivity and accuracy of current primer probe detection need to be improved.

Method used

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  • Novel coronavirus detection kit and detection method thereof
  • Novel coronavirus detection kit and detection method thereof
  • Novel coronavirus detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] The preparation of each reagent of embodiment 1

[0093] Use a DNA synthesizer to synthesize the following sequences by the solid-phase phosphoramidite triester method:

[0094] Primer probe set Covid-19A: containing ORF1ab-F, ORF1ab-R, ORF1ab-P;

[0095] Primer probe set Covid-19B: including N-F, N-R, N-P; primer probe set Covid-19C: including E-F, E-R, E-P; internal standard primer probe set RP TEST: including RP-F, RP-R, RP -P; Positive control substance: artificially synthesized gene containing ORF1ab, N and E gene sequences of novel coronavirus;

[0096] Negative control substance: TE buffer solution (PH=8.0), purchased from Beijing Solaibao Technology Co., Ltd.;

[0097] Reaction Buffer: containing Tris buffer, magnesium ions, dA / G / C / UTPs, DNA polymerase, reverse transcriptase, UNG enzyme: purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0098] The probe ORF1ab-P is labeled with FAM, the probe N-P is labeled with VIC, the probe E-P is labeled with CY...

Embodiment 2

[0099] Example 2 Kit with ORF1ab gene and N gene as detection target

[0100] Prepare reagents according to the composition of the following table:

[0101] component name main ingredient PCR reaction solution Reaction Buffer, dNTPs, Taq enzyme, UDG enzyme PCR reverse transcriptase RT enzyme PCR primer probe Primers and probes for ORF1ab and N genes; primers and probes for internal standard RP genes positive control Pseudovirus containing ORF1ab and N gene fragments negative control Pseudovirus containing internal standard RP gene fragment

[0102] The primer preparation concentration was 0.4 μmol / L, and the probe preparation concentration was 0.2 μmol / L. 0.1mmol / L dATP, 0.1mmol / L dCTP, 0.1mmol / L dGTP, 0.1mmol / L dUTP, 1mmol / L MgCl 2 , Enzyme-free water with a volume percentage of 30%; 5 U of reverse transcriptase, 10 U of RNase inhibitor, 1 U of hot-start DNA polymerase, and 1 U of thermosensitive UDG enzyme.

[0103] 1. Sam...

Embodiment 3

[0135] Example 3 Kit with the ORF1ab gene as the detection target

[0136] According to the method described in Example 2, a kit was prepared and genetic testing was performed. It differs only in:

[0137] Prepare reagents according to the composition of the following table:

[0138] component name main ingredient PCR reaction solution Reaction Buffer, dNTPs, Taq enzyme, UDG enzyme PCR reverse transcriptase RT enzyme PCR primer probe ORF1ab gene primers, probe set; internal standard RP gene primers, probe set positive control Pseudovirus containing ORF1ab gene fragment negative control Pseudovirus containing internal standard RP gene fragment

[0139] The primer preparation concentration was 0.4 μmol / L, and the probe preparation concentration was 0.2 μmol / L. 0.1mmol / L dATP, 0.1mmol / L dCTP, 0.1mmol / L dGTP, 0.1mmol / L dUTP, 1mmol / L MgCl 2 , Enzyme-free water with a volume percentage of 30%; 5 U of reverse transcriptase, 10 U o...

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Abstract

The invention provides a novel coronavirus detection kit and a detection method thereof, and belongs to the technical field of molecular biological detection. A series of primer probe groups for detecting the novel coronavirus are redesigned, an existing detection method is improved, and detection targets are further increased, so that the detection sensitivity of the novel coronavirus is improved, false negative results can be remarkably reduced especially for detection of low-copy-number samples, the sensitivity and the accuracy of detection are effectively improved, and high-efficiency, high-specificity and low-cost detection is realized. The kit can be used for qualitatively detecting the novel coronavirus genes in pneumonia suspected cases and suspected aggregated case patients infected by the novel coronavirus in vitro and samples such as nasopharyngeal swabs and sputum of other patients needing novel coronavirus infection diagnosis or differential diagnosis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a novel coronavirus detection kit and a detection method thereof. Background technique [0002] The 2019 novel coronavirus (COVID-19) belongs to the genus Betacoronavirus and is the seventh human-infecting coronavirus (HCoV) identified in human history. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV . On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) announced that the classification of the 2019 novel coronavirus was named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2). The disease caused by viral infection is officially named COVID-19. The 2019 novel coronavirus pneumonia is mainly transmitted through droplets and contact, and the crowd is generally susceptible. The general symptoms of 2019-nCoV infection are: fever, fatigue, dry cough, and gradual dyspnea. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686Y02A50/30
Inventor 韩亚平柴旭阳臧百盛张淼顾城玮
Owner 北京吉检医疗科技有限公司
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