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Crimean-Congo hemorrhagic fever virus detection method and primer probe set

A Congo hemorrhagic fever, primer probe technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of Crimea-Congo hemorrhagic fever virus detection

Active Publication Date: 2020-10-30
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of this disclosure is to solve the defects in the prior art that Crimea-Congo hemorrhagic fever virus cannot be detected rapidly, sensitively and specifically, and provide a detection primer probe and kit, and established a rapid, sensitive, specific and simple detection method for Crimea-Congo hemorrhagic fever virus based on Recombinase Polymerase Amplification (RPA) technology

Method used

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  • Crimean-Congo hemorrhagic fever virus detection method and primer probe set
  • Crimean-Congo hemorrhagic fever virus detection method and primer probe set
  • Crimean-Congo hemorrhagic fever virus detection method and primer probe set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This example is used to illustrate the verification test of the Crimean-Congo hemorrhagic fever virus specific primer probe.

[0059] A reagent company was commissioned to synthesize the Crimean-Congo hemorrhagic fever virus primer probe set and internal quality control primer probe set shown in Table 2.

[0060] Table 2

[0061]

[0062] In the nucleotide sequence shown in SEQ ID NO.3, the 31st base from the 5' end is labeled with a FAM luminescent group, the 32nd base is connected with an abasic site, and the 34th base is marked with a quencher The killing group BHQ1, the 3' end is labeled with a phosphate group.

[0063] In the nucleotide sequence shown in SEQ ID NO.6, the 31st base from the 5' end is labeled with a CY5 luminescent group, the 32nd base is connected with an abasic site, and the 34th base is marked with a quencher The killing group BHQ3, the 3' end is labeled with a phosphate group.

Embodiment 2

[0087] Construction of primers, probes and kits for detection of Crimean-Congo hemorrhagic fever virus by recombinant enzyme polymerase amplification

[0088] The kit contains 2×RT-RPA buffer, lyophilized enzyme powder, 2 μL (10 μmol / L) of each primer, 0.5 μL (10 μmol / L) of probe, 1 μL (200 U / μL) of reverse transcriptase, positive control, ultra pure water.

[0089] The reaction system detected by the kit is 50 μL, which is prepared as follows: 25 μL of 2×RT-RPA buffer; lyophilized enzyme powder; 2 μL of each primer (10 μmol / L), 0.5 μL of probe (10 μmol / L); 1 μL of reverse transcriptase (200U / μL); template 5 μL, ultrapure water 15 μL.

Embodiment 3

[0091] Kit operation and result judgment

[0092] 1. Extraction of viral nucleic acid

[0093] In vitro transcribed RNA was extracted using commercially available extraction reagents.

[0094] 2. Preparation of reaction system

[0095] Take a 200 μL RPA tube to prepare a 50 μL reaction system, which is prepared as follows: 25 μL of 2×RT-RPA buffer; lyophilized enzyme powder; 2 μL of each primer (10 μmol / L), 0.5 μL of probe (10 μmol / L); reverse transcription Enzyme 1 μL (200U / μL); template 5 μL, ultrapure water 15 μL.

[0096] 3. RPA response

[0097] Put the RPA tube into the real-time fluorescence RPA instrument, select FAM and CY5 as the reporter group, and the RPA reaction program is as follows: 39°C for 10s, 39°C for 10s, 39°C for 10s (collecting fluorescence), 40 cycles.

[0098] 4. Result judgment

[0099]Blank control and negative and positive controls are established, otherwise the experimental results will be deemed invalid. If the sample has an amplification cu...

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Abstract

The present disclosure discloses a primer probe set and a kit using recombinase polymerase amplifying for detecting Crimean-Congo hemorrhagic fever (CCHF) virus and a detection method. The primer probe set includes primers with nucleotide sequences shown in SEQ ID NO. 1 to 2 and a probe with a nucleotide sequence shown in SEQ ID NO.3. The present disclosure also provides the kit using recombinasepolymerase amplifying for detecting the Crimean-Congo hemorrhagic fever. The kit includes the primer probe set. The primer probe set and the kit significantly improve the sensitivity, specificity, andsimplicity of the detection of the Crimean-Congo hemorrhagic fever virus.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular to a detection method for Crimean-Congo hemorrhagic fever virus and a primer probe set. Background technique [0002] Crimean-Congo hemorrhagic fever is caused by Crimean-Congo hemorrhagic fever virus (Crimean-Congo hemorrhagic fever virus). It is an acute viral infectious disease with a high fatality rate transmitted by ticks. Tick-borne natural focal viral diseases are distributed in Europe, Asia and Africa. It was first discovered in China and Xinjiang Bachu, so it is also called Xinjiang hemorrhagic fever in China. [0003] Crimean-Congo hemorrhagic fever is currently the only biosafety level 4 (Laboratory Biosafety Level 4, BSL-4) pathogen that has been confirmed to have a natural outbreak in my country. Several local outbreaks have occurred in Xinjiang. The disease is characterized by hemorrhage of the skin, mucous membranes, and internal organs. The clinical manifestations ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2521/507C12Q2521/101C12Q2521/107
Inventor 杨海英王雷张志强
Owner 北京卓诚惠生生物科技股份有限公司
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