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Multiple primers and kits for rapid detection of influenza A, influenza B and novel coronavirus

A technology of influenza B virus and influenza A virus, which is applied in the field of molecular biology detection technology and molecular diagnosis, can solve the problems of long time consumption and high requirements of laboratory environment, so as to avoid cross-infection, improve detection efficiency and high sensitivity Effect

Active Publication Date: 2021-06-18
广州领上源生物科技有限公司
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] At present, most of the new coronavirus nucleic acid detection products or multiple detection products of new coronavirus and influenza virus on the market are ordinary fluorescent PCR detection products, which take a long time. Except for nucleic acid extraction, the amplification detection process basically takes 1.5 to 2 hours, and it is very difficult for the experiment. Room environment requirements are high

Method used

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  • Multiple primers and kits for rapid detection of influenza A, influenza B and novel coronavirus
  • Multiple primers and kits for rapid detection of influenza A, influenza B and novel coronavirus
  • Multiple primers and kits for rapid detection of influenza A, influenza B and novel coronavirus

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Embodiment 1

[0059] Design and optimization of specific primer probes.

[0060] Referring to the influenza diagnosis and treatment guidelines and the new coronavirus diagnosis and treatment guidelines, nucleic acid testing is the gold standard for judging the diagnosis of new coronavirus infection. In order to ensure the detection of influenza A virus, influenza B virus and new coronavirus, according to the nucleic acid sequences of these three pathogens , analyzed the conserved regions of their genes, and finally selected primer probes for the conserved regions of the M gene of influenza A virus, the NS gene of influenza B virus, and the N gene of the new coronavirus, in order to obtain multiplex genes with better specificity and sensitivity. Primer-probe system combination, designed a total of 60 primers and 28 probes, and combined multiple experiments. At the same time, the human reference gene was designed to monitor the sample collection, nucleic acid extraction process and PCR amplif...

Embodiment 2

[0079] A kit for multiple rapid detection of influenza A virus, influenza B virus and novel coronavirus, comprising:

[0080] Reagent Ⅰ (735μl): Taq polymerase (0.04U / uL), MgCl 2 (4.5mM), dNTPs (0.2mM), and the primer concentrations of influenza A virus, influenza B virus and novel coronavirus are all 0.4μM, and the probe concentration is 0.25μM.

[0081] Reagent II (1500U): fast reverse transcriptase in freeze-dried form.

[0082] Negative quality control: normal saline.

[0083] Positive quality control product: pseudovirus containing influenza A virus, influenza B virus, new coronavirus and internal reference gene target fragment, the concentration of pseudovirus is 1.0×10 2 ~1.0×10 4 copies / μL.

Embodiment 3

[0085] The detection method of the kit for multiple rapid detection of influenza A virus, influenza B virus and novel coronavirus.

[0086] 1. Specimen type: oropharyngeal swab, nasopharyngeal swab.

[0087] 2. Nucleic acid extraction:

[0088] Use commercial RNA extraction kits, such as nucleic acid extraction reagents based on silica gel membrane spin column method or nucleic acid extraction reagents based on magnetic bead method, and operate according to the kit instructions, and finally collect 80 μl RNA solution for direct detection or storage in -80°C. Both negative quality control and positive quality control need to be extracted.

[0089] 3. Amplification reaction:

[0090] Each tube of a complete detection system should include 14.7 μL reagent I, 0.3 μL reagent II (in freeze-dried form, add 20 μL depc water to dissolve before use, and place it on ice for more than 10 minutes before use), 5 μL quality control material or nucleic acid of the sample to be tested . S...

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Abstract

The invention provides a multiple primer and kit for rapid detection of influenza A, influenza B and novel coronavirus, and relates to the field of molecular biology detection technology and molecular diagnosis. The method of the present invention is based on the method of reverse transcription thermal convection real-time fluorescent PCR, a specific multiple rapid diagnostic system developed for the respective conserved regions of influenza A virus, influenza B virus and new coronavirus, and also detects human endogenous genes at the same time. Sample quality control. The multiple primers and kit have good specificity and high sensitivity, and are also simple, fast and practical, meeting the detection needs of influenza A virus, influenza B virus and new coronavirus in entry-exit and on-site environments.

Description

technical field [0001] The invention relates to the field of molecular biology detection technology and molecular diagnosis, in particular to a multiple primer and kit for rapid detection of influenza A, influenza B and novel coronavirus. Background technique [0002] Novel coronavirus pneumonia is an acute infectious pneumonia, and its pathogen is a new type of coronavirus, which was named "2019-nCoV" by the World Health Organization on January 12, 2020. The virus is highly contagious. After infecting people, the initial symptoms are mostly fever, fatigue and dry cough, and gradually develop severe manifestations such as dyspnea. Most of the prognosis is good, but some severe cases can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even Therefore, accurate detection of infected persons, carriers and suspected populations is crucial to the prevention and control of the epidemic. [0003] Influenza viruses (Flu), also referred to as influenza viruse...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2561/113C12Q2563/107C12Q2537/143C12Q2521/107Y02A50/30
Inventor 曹艳高秀洁梁建芳蔡婷婷张晓刚陈廷友
Owner 广州领上源生物科技有限公司
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