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Novel coronavirus(2019-nCoV) ORF1ab gene nucleic acid detection kit

A coronavirus and kit technology, applied in the field of biotechnology and molecular diagnosis, can solve the problems of high cost, long time-consuming 2019 novel coronavirus, failure to form a rapid and complete 2019 novel coronavirus detection system, etc., and achieve high specificity Effect

Active Publication Date: 2020-04-17
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the traditional isolation and culture method for culturing and detecting 2019-nCoV is time-consuming and costly, and has failed to form a rapid and complete 2019-nCoV detection system

Method used

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  • Novel coronavirus(2019-nCoV) ORF1ab gene nucleic acid detection kit
  • Novel coronavirus(2019-nCoV) ORF1ab gene nucleic acid detection kit
  • Novel coronavirus(2019-nCoV) ORF1ab gene nucleic acid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106]Example 1 Novel coronavirus ORF1ab gene detection kit

[0107] The kit consists of PCR reaction solution, PCR enzyme system, internal standard, positive control substance, negative control substance and DEPC water. The PCR reaction solution consists of 5×RT-PCR Buffer, primers and probes, and dNTPs; the PCR enzyme system It consists of reverse transcriptase, RNase inhibitor, hot-start DNA polymerase and enzyme diluent; the positive control is prepared from a pseudovirus containing the ORF1ab target sequence, the negative control is normal saline, and the internal standard is containing the internal target Sequenced pseudoviruses were prepared.

[0108] The final concentration of the primer in the amplification system is 800nM, and the final concentration of the probe is 200nM.

[0109] The reaction system of the kit is 25 μL, specifically as follows: 3 μL of PCR enzyme system, 17 μL of PCR reaction solution, 2-5 μL of nucleic acid template, and supplemented with DEPC wa...

Embodiment 2

[0110] Example 2 The operation and result determination of the kit

[0111] (1) Extraction of viral genomic DNA

[0112] Use nucleic acid extraction or purification reagents (product number: DA0623) from Sun Yat-Sen University Daan Gene Co., Ltd. to extract nucleic acids in the sample processing area according to the instructions. Before extraction, each sample was extracted by adding an internal standard at a ratio of 50:1. Both the negative control substance and the positive control substance are involved in the extraction, as the quality control of the environment and PCR detection reagents.

[0113] (2) Preparation of reaction system

[0114] The kit of Example 1 was used to carry out the following experiments. After the PCR reaction solution of the kit was completely dissolved at room temperature, it was shaken and mixed quickly. The 25 μL PCR reaction system was: 17 μL of PCR reaction solution, 3 μL of enzyme system, and 5 μL of template.

[0115] (3) PCR amplificatio...

Embodiment 3

[0128] Example 3 Kit Specific Detection

[0129] The kit described in Example 1 is used for influenza A H3N2, H7N9, influenza B Yamagata, respiratory syncytial virus type A, adenovirus type 1, type 3, enterovirus type A, human metapneumovirus, Epstein-Barr virus, Human cytomegalovirus, rotavirus, norovirus, Mycoplasma pneumoniae, Mycobacterium tuberculosis, coronavirus (HKU1, OC43, NL63, 229E), SARS pseudovirus, MERS pseudovirus) and novel coronavirus (2019-nCoV) There was no detection of FAM channel fluorescence during the detection; however, the positive samples for the novel coronavirus were detected as positive. It shows that the specificity of the kit of the present invention is good.

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Abstract

The invention provides a novel coronavirus ORF1ab gene nucleic acid detection kit, and particularly discloses a kit for detecting the ORF1ab gene of the novel coronavirus and a method thereof, which have extremely high sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology and molecular diagnosis, in particular, the invention relates to a nucleic acid detection kit for novel coronavirus ORF1ab gene. Background technique [0002] Since December 2019, a number of cases of pneumonia of unknown cause have been discovered one after another, and it has been confirmed as an acute respiratory infectious disease caused by a new type of coronavirus infection. Based on the current epidemiological investigation, the incubation period is generally 3-7 days, and the longest does not exceed 14 days. The clinical manifestations of this epidemic are mainly fever, fatigue, and dry cough. A small number of patients have symptoms such as nasal congestion, runny nose, and diarrhea. Some patients only showed low-grade fever, mild fatigue, etc., without pneumonia, and mostly recovered after 1 week. The World Health Organization named the pathogen causing this case of viral pneumonia as the...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/101
Inventor 蒋析文彭海龙范建
Owner DAAN GENE CO LTD
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