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Novel coronavirus nucleic acid chromatography detection kit and application

A detection kit and coronavirus technology, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, DNA/RNA fragments, etc., which can solve the problems of the possibility of aerosol pollution, difficult DNA degradation, and low specificity , to achieve the effects of low equipment requirements, easy degradation, and low possibility of pollution

Inactive Publication Date: 2021-04-13
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the technical problems of the novel coronavirus detection kit in the prior art, which takes a long time to detect, requires special equipment, DNA is not easily degraded and easily polluted, and has low sensitivity and specificity, this application provides a novel coronavirus nucleic acid chromatography detection kit And application, no RNA extraction process, no need for PCR equipment, based on RNA constant temperature amplification, and the amplified product is RNA, easy to degrade, and the possibility of aerosol pollution is extremely low; specially designed primers and probes have high sensitivity and specificity The advantages of strong stability and simple operation

Method used

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  • Novel coronavirus nucleic acid chromatography detection kit and application
  • Novel coronavirus nucleic acid chromatography detection kit and application
  • Novel coronavirus nucleic acid chromatography detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Preparation of nucleic acid detection test strips

[0053] The main raw materials needed in the preparation of nucleic acid detection test strips: nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC bottom plate, etc.

[0054] 1. Spray film:

[0055] Detection line N-T line: N gene coated probe sequence modified by biotinylation at the 5' end, (20μM), spray volume: 2-3μL / cm;

[0056] Detection line ORF1ab-T line: 5' end biotinylated modified ORF1ab gene coating probe sequence, (20μM), spray volume: 2-3μL / cm;

[0057] Internal reference of detection line-T line: ACTB gene-coated probe sequence modified by biotinylation at the 5' end, (20 μM), spray volume: 2-3 μL / cm;

[0058] Quality control line (line C): biotinylated modification at the 5' end, capable of capturing nano-gold probes (20 μM), spray volume: 2-3 μL / cm.

[0059] After spraying the film, put it in a clean incubator at 37°C to dry for 2 hours, and store it in a dry environment for later...

Embodiment 2

[0063] Sensitivity test comparison test test

[0064] The colloidal gold prepared by this development method and the method published in Chinese patent application CN111455099A was used for a sensitivity comparison test. Select a known concentration of the pseudovirus containing the 2019-nCoV target gene, perform a 10-fold concentration gradient dilution, repeat each gradient 3 times, and use the lowest dilution concentration with a 100% positive detection rate as the estimated detection limit, the estimated detection limit After determination, the 2019-nCoV pseudovirus was diluted to near the estimated detection limit concentration, and tested with a kit, and each concentration was tested 20 times to further accurately determine the lowest detection limit concentration (select the dilution with a positive rate of more than 95%) degree as the detection limit sensitivity of this kit).

[0065] Table 2 Determination of the detection limit of 2019-nCoV

[0066]

[0067]

...

Embodiment 3

[0070] specificity verification

[0071] Other pathogens that are similar in species to 2019 novel coronavirus or cause similar symptoms, such as seasonal influenza A H1N1 virus, novel influenza A H1N1(2009) virus, influenza A H3N2, H7N9, influenza B Yamagata, influenza B Victoria, Respiratory Syncytial Virus Type A, Respiratory Syncytial Virus Type B, Parainfluenza Type I, Parainfluenza Type II, Parainfluenza Type III, Rhinovirus Groups A, B, C, Adenovirus Type 1, 7, Enterovirus A , B, C, D types, rotavirus group A, norovirus type GI, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, coronavirus (HKU1, OC43, NL63, 229E ), a MERS coronavirus pseudovirus, a cross-reaction test was carried out to verify the specificity of the kit’s detection. The results showed that there was no cross-reaction between the kit and other microorganisms, reflecting the strong specificity of the kit’s detection of pathogens.

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Abstract

The invention relates to the technical field of gene detection, in particular to a novel coronavirus nucleic acid chromatography detection kit and application. Aiming at the technical problems that a novel coronavirus detection kit is long in detection time, needs special equipment and is low in sensitivity and is not high in specificity and DNA is not easy to degrade and is easily polluted in the prior art, the novel coronavirus nucleic acid chromatography detection kit and the application are provided, an RNA extraction process and PCR instruments are not needed, the kit is based on RNA isothermal amplification, moreover, an amplification product is RNA and is easy to degrade, and the possibility of aerosol pollution is extremely low; and specially designed primers and probes have the advantages of high sensitivity, strong specificity and simple operation.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a novel coronavirus nucleic acid chromatography detection kit and its application. Background technique [0002] The 2019-nCoV belongs to the genus β-coronavirus, which has an envelope, and the particles are round or oval, with a diameter of 60-140nm. The main clinical manifestations are fever, dry cough, and fatigue. Severe patients gradually develop more serious diseases such as dyspnea and / or hypoxemia; severe patients rapidly progress to acute respiratory distress syndrome, septic shock, and metabolic acidity that is difficult to correct. Toxicity and coagulation disorders. The novel coronavirus is an RNA positive-strand virus with a sequence similarity of 79.6% to the SARS coronavirus. On February 11, 2020, the International Committee on Virological Classification tentatively named the novel coronavirus as SARS-CoV-2. [0003] Accurate and rapid identification of S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166Y02A50/30
Inventor 刘文干朱丽君韦玉军王娟桂林李成俊
Owner 安徽安龙基因科技有限公司
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