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PCR-based method for detecting viruses through nucleic acid amplification and kit

A virus, dimethyl sulfoxide technology, applied in the field of molecular biology, can solve the problems of complex, error-prone, cumbersome detection methods, etc., and achieve the effects of high virus detection, prevention of RNA degradation, and high sensitivity

Pending Publication Date: 2020-11-24
JIANGSU COWIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In general, the above-mentioned traditional detection methods are cumbersome, complex and error-prone
Their inefficiency can severely limit society's ability to respond quickly and effectively to emergencies such as outbreaks of the SARS-CoV-2 virus, and may lead to undesirable delays in isolating and treating infected patients

Method used

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  • PCR-based method for detecting viruses through nucleic acid amplification and kit
  • PCR-based method for detecting viruses through nucleic acid amplification and kit
  • PCR-based method for detecting viruses through nucleic acid amplification and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Comparison of different virus preservation solutions

[0044] With IBV virus (chicken infectious bronchitis virus, a kind of coronavirus) as a reference substance, the infectious bronchitis virus of different dilutions is preserved in different virus preservation solutions, and then these samples are divided into two parts respectively: one One was used for direct PCR detection, and the other was used to extract viral RNA and then perform PCR detection on the RNA. We compared the preservation effect and sensitivity of different preservation solutions.

[0045] Material:

[0046] We tested and compared the following preservation solutions:

[0047] Preservation solution A: NaOH (20mmol / L), surfactant (0.5% [w / v]), DMSO (5% [v / v]); sucrose (8% [w / v]), Chelex-100 ( 0.9% [w / v]).

[0048] Preservation solution B: β-mercaptoethanol (10mmol / L-15mmol / L), 8-hydroxyquinoline (0.05mol / L-0.1mol / L, sodium acetate (2mol / L-5mol / L), dodecyl Sodium sarcosine (5%-10% [...

Embodiment 2

[0072] Example 2: Viral nucleic acid detection kit and qPCR amplification

[0073] This embodiment provides a nucleic acid amplification detection kit for a new coronavirus (SARS-CoV-2) sample, said kit comprising (1) SARS-CoV-2 preservation solution, (2) SARS-CoV-2 PCR Amplification reaction lyophilized powder, (3) positive control; and (4) negative control.

[0074] Material preparation:

[0075] (1) SARS-CoV-2 preservation solution (pH 8.0) was prepared according to the following table:

[0076] Table 6

[0077] Reagent Final concentration NaOH 10–200mM Tris.HCl 10–200mM KCl(2M) 10–100mM MgCl 2 (1M)

1.5–6mM DMSO 1–5% (v / v) glycerin 1–10% (v / v) BSA 1–5ug / ul fish gelatin 0.1–1% (w / w)

[0078] (2) SARS-CoV-2 nucleic acid amplification reaction lyophilized powder:

[0079]According to the RNA sequence design of SARS-CoV-2 virus ORF1ab and N gene, primers and probes are as follows:

[0080] ORF1ab-F...

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Abstract

The invention provides a virus preservation solution, a nucleic acid amplification reaction preparation (which can be in the form of freeze-dried powder), a qPCR-based method for detecting viruses through nucleic acid amplification, and a PCR kit. The virus preservation solution can preserve a virus sample for a long time at room temperature and prevent RNA degradation. The sample solution can bedirectly mixed with a nucleic acid amplification reaction preparation to be used for qPCR amplification, a virus nucleic acid extraction step is not needed, and viruses can be rapidly detected with high sensitivity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a virus preservation solution, a nucleic acid amplification reaction solution, a nucleic acid PCR amplification method, and a PCR amplification kit. Background technique [0002] At present, the commonly used viral RNA detection method usually includes three steps: 1) collect the subject’s virus sample and transfer the sample to the laboratory; 2) extract the viral RNA from the virus sample by silica gel membrane column or magnetic bead adsorption; 3 ) for RT-PCR amplification and fluorescence quantitative detection (results can be interpreted based on Ct values). [0003] In this method, RNA extraction kits are usually separated from virus sample preservation kits and RT-qPCR detection kits. In addition, the collected samples often contain RT-PCR polymerase inhibitors, which will hinder nucleic acid amplification if directly mixed with PCR reagents. In addition, under normal c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N7/00C12R1/93
CPCC12Q1/701C12Q1/686C12N7/00C12N2770/20011C12Q2521/107C12Q2521/101C12Q2527/125C12Q2531/113C12Q1/70C12Q1/6806C12Q1/6851
Inventor 王春香郭金海肖欢欢
Owner JIANGSU COWIN BIOTECH CO LTD
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