Marburg virus detection primer, probe and kit

A Marburg virus, primer-probe technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as inability to detect Marburg virus sensitively and specifically, and reduce labor costs. and time cost effect

Inactive Publication Date: 2018-04-20
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 Marburg virus cannot be detected quickly, sensitively and specifically, to provide a detection primer probe and kit for Marburg virus, and to establish a method based on recombinase polymerase amplification. Rapid, sensitive, specific and simple detection method of Marburg virus by Recombinase Polymerase Amplification (RPA) technology

Method used

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  • Marburg virus detection primer, probe and kit
  • Marburg virus detection primer, probe and kit
  • Marburg virus detection primer, probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The synthesis of embodiment 1 primer and probe

[0053] A reagent company was commissioned to synthesize the primer-probe sets shown in Table 2.

[0054] Table 2

[0055]

[0056] The nucleotide sequences of the upstream and downstream primers and probes for detecting Marburg virus are respectively shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0057] In the nucleotide sequence shown in SEQ ID NO.3, the 19th base from the 5' end is labeled with a FAM luminescent group, the 20th base is connected with an abasic site, and the 22nd base is labeled with a quencher Killing group BHQ1.

[0058] The nucleotide sequences of the upstream and downstream primers and probes for detecting the internal quality control are respectively shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0059] In the nucleotide sequence shown in SEQ ID NO.6, the 31st base from the 5' end is marked with a VIC luminescent group, the 32nd base is connected with an abasic site, and the 34th...

Embodiment 2

[0060] Example 2 specificity verification

[0061] The Marburg virus-specific primer pair and probe designed in Example 1 were evaluated and verified, mainly evaluating specificity, minimum detection limit and coverage.

[0062] Specificity evaluation: Select in vitro transcription of Hantaan virus, Seoul virus, Andes virus, Sindbis virus, dengue virus, Ebola virus, Lassa virus nucleic acid as the specificity evaluation nucleic acid. The total nucleic acid concentration of each sample was 0.001 ng / μL, and the nucleic acid of each sample was mixed as a template for specificity evaluation, and RPA amplification was performed.

[0063] Preparation of RPA reaction system: 50 μL of total system, 29.5 μL of rehydration buffer, 2.5 μL of magnesium acetate solution (280 mmol / L), primer 2 μL (10 μM) each, 0.5 μL (10 μM) probe, 1 μL reverse transcriptase (200 U / μL), 5 μL template, and make up the rest with water.

[0064] The reaction conditions of the RPA reaction: FAM was selected a...

Embodiment 3

[0067] Embodiment 3 minimum detection limit verification

[0068] Evaluation of the minimum detection limit: the standard plasmid with the recombinant Marburg virus gene sequence was cut with restriction enzymes SpeI and PvuII, linearized and transcribed in vitro; the initial concentration was selected as 10 5 copy / μL transcript, which was serially diluted to 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 copies / μL, 2 copies / μL and 1 copy / μL. System preparation and RPA reaction conditions are the same as those in Example 2 for specificity evaluation. In the reaction system, the concentration of the virus was 5×10 4 copy / system, 5×10 3 copy / system, 5×10 2 copies / system, 50 copies / system, 10 copies / μL and 5 copies / system.

[0069] Judgment of RPA reaction results: blank control and negative control are established, otherwise the experimental results will be invalid; if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.1-2 and the probe of SEQ ID...

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Abstract

The present disclosure discloses a primer probe set and a kit using recombinase polymerase amplifying for detecting Marburg 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 recombinase polymerase amplifying for detecting the Marburg virus. The kit includes the primer probe set. The primer probe set and the kit significantly improve the sensitivity, specificity, and simplicity of the detection of the Marburg virus.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular to a primer probe set, a kit and a detection method for detecting Marburg virus. Background technique [0002] Marburg hemorrhagic fever is caused by Marburg virus (MARV), which can be transmitted through close contact between people. It is highly contagious and has a high fatality rate. Since the first outbreak and confirmation of Marburg hemorrhagic fever in 1967, there have been 40 years of epidemic history, during which there were 3 large-scale outbreaks. Marburg hemorrhagic fever, with its highly contagious outbreak and high lethality, makes people pay more and more attention to the potential threat of Marburg hemorrhagic fever to human health and public health security. [0003] The initial symptoms of Marburg hemorrhagic fever are similar to malaria, SARS, influenza or yellow fever, etc., and the late symptoms are similar to Ebola hemorrhagic fever, Lassa fever, hemorrhagic fev...

Claims

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Application Information

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