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Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescent RPA

A coronavirus and probe technology, applied in the field of applied biology, can solve the problem of not particularly good timeliness, and achieve the effect of low detection sensitivity and wide application prospects.

Active Publication Date: 2020-06-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the national CDC and WHO both recommend fluorescent quantitative PCR as a diagnostic method for the new coronavirus. However, the timeliness in the actual application process is not particularly good. The reverse transcription fluorescent PCR takes 90 minutes, and a special fluorescent PCR instrument is needed for data analysis.

Method used

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  • Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescent RPA
  • Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescent RPA
  • Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescent RPA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the acquisition of primer, probe sequence and the construction of positive plasmid

[0047] According to the full-length nucleic acid sequence of the new coronavirus (SARS-CoV2), compare and analyze it, and compare it with the full-length sequence of SARS virus and SARS-like virus to find out the SARS-CoV2 virus-specific sequence S gene and virus conserved sequence N gene, SARS-like virus conservative sequence E gene, Orf1Ab gene, M gene, then design primers and probes according to the above sequence; the primers and probes involved in this application are shown in the following table:

[0048]

[0049]

[0050] This application completes the sensitivity test of the fluorescent RPA method by constructing a standard plasmid. Based on the pUC57 plasmid, the target gene was synthesized and the recombinant plasmid was constructed. Transform Escherichia coli (Escherichia coli) competent cells by heat shock method, smear a plate to select monoclonal colon...

Embodiment 2

[0051] Embodiment 2, the processing of actual detection sample and virus RNA genome extraction

[0052] Test samples include but are not limited to human serum, plasma, whole blood, feces, throat swabs, and sputum. Sample processing: Dissolve 1g of feces or a patient's throat swab in 1ml of normal saline, shake and mix well, centrifuge at 5000r / min for 10min, and take the supernatant to extract viral RNA. Add an equal amount of 10g / L acetylcysteine ​​to the sputum, shake it at room temperature for 1 hour, and extract viral RNA after fully liquefied. Anticoagulants (heparin, etc.) should be added to plasma and whole blood.

[0053] The guanidinium isothiocyanate-glass powder method extracts viral RNA: the specific operation steps are as follows: (1) add guanidine isothiocyanate-glass powder lysate (4.7mol / L guanidine isothiocyanate, 20mmol / LEDTA, 100mmol / L Tris-HCl pH6.4, 1% Triton X-100 and glass powder 10g) 0.9ml, mix well, act at room temperature for 10min, centrifuge at ...

Embodiment 3

[0055] Embodiment 3, implementing triple fluorescent RPA

[0056] The triple fluorescent RPA reaction includes two steps: system preparation, amplification and signal reading. Use the 50 μL system recommended by the RNA Constant Temperature Rapid Amplification Kit (fluorescent type), use the prepared RNA or (DNA plasmid) as a template, and use the primers and probe sequences shown in the abstract to perform a triple fluorescent RPA reaction. Fluorescent RPA reaction system (50 μL): Buffer A29.4 μL, upstream primers NF, EF, SF each 0.8 μL (10 μmol / L), downstream primers NR, ER, SR each 0.8 μL (10 μmol / L), probes NP, EP , SP 0.3 μL each (10 μmol / L), nucleic acid template 2 μL, RNase-free Water 10.2 μL, ROX dye 0.2 μL, Buffer B 2.5 μL. The reaction conditions were set as follows: 42° C., 20 min, and the fluorescence signal was collected every 30 s, for a total of 40 cycles. The instrument automatically collects fluorescence signals, and can observe fluorescence amplification si...

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Abstract

The invention discloses a triple RPA primer, probe, identification method and identification kit for rapidly screening and detecting a novel coronavirus (SARS-CoV2) and other SARS-similar coronaviruses. A first set of primers and probes (marked as FAM) are designed in a region of an N gene sequence of the SARS-CoV2 virus; a second set of primers and probes (marked as HEX) are designed in an E genesequence in a conserved region of an SARS-similar virus; and a third set of primers and probes (marked as Cy5, being different from the other SARS-similar coronaviruses) are designed in a specific region of an S gene sequence of the SARS-CoV2 virus. Compared with qPCR nucleic acid detection methods recommended, by the WHO and the national CDC, to be used in novel coronavirus clinics, the triple RPA primer, the probe, the identification method and the identification kit have the advantages that the sensitivity is high, the detection speed is high, and the novel coronavirus and the other SARS-similar coronaviruses can be effectively distinguished; and the nucleic acid detection time limit of the virus can be shortened from 90 minutes to 20 minutes, and rapid screening demands on the novel coronavirus and the other SARS-similar coronaviruses can be satisfied.

Description

technical field [0001] The invention belongs to the field of applied biotechnology, and in particular relates to specific rapid screening and detection primers, probes, identification methods and identification kits for screening and identifying novel coronaviruses and other SARS-like coronaviruses. Background technique [0002] The pneumonia epidemic (COVID-19) caused by the new coronavirus (SARS-CoV2) is currently sweeping the world, and it has a huge impact on the economy, society, and people's normal life, and the rapid diagnosis of new coronavirus cases has become the key to cutting off the source of infection and controlling the epidemic. important means of communication. In particular, the current domestic epidemic situation is stabilizing, the foreign epidemic situation is severe, and imported cases are increasing, which requires higher accuracy in nucleic acid screening. [0003] At present, the national CDC and WHO both recommend fluorescent quantitative PCR as a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/16C12Q2531/119C12Q2537/143C12Q2563/107Y02A50/30
Inventor 吕继洲张舟袁向芬林祥梅吴绍强王彩霞邓俊花冯春燕
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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