RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit

An Ebola virus and kit technology, which is applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effects of strong specificity, shortened detection time, and high sensitivity

Inactive Publication Date: 2016-03-16
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to determine the appropriate method for Ebola virus, technicians still need to do a lot of in-depth research, and there are no clear ideas and results that can be used for reference.

Method used

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  • RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit
  • RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit
  • RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, Ebola virus RPA primer and probe design and screening

[0044] (1) Design of primers and probes

[0045] The inventor analyzed and determined through literature search that the conserved sequence in the Ebola virus NP gene is used as the target gene in the present invention. The known target gene sequence, ie the oligonucleotide sequence shown in SEQ ID NO.: 1, was obtained from the NCBI database. According to the RPA primer and probe design principles, 8 primers and 1 probe were designed, as shown in Table 1.

[0046] Table 1 Primers and probes

[0047]

[0048] (2) Primer screening

[0049] Artificially synthesize a positive plasmid containing the Ebola NP gene sequence, use this plasmid as a template, comprehensively combine primers and probes, and perform RPA detection respectively, and use the cumulative fluorescence intensity of the reaction and detection time as indicators to screen out the sensitivity The highest primer-probe combination for...

Embodiment 2

[0059] Embodiment 2: Sensitivity evaluation of RPA detection

[0060] The positive plasmid was serially diluted 10 times to 10 7 To a series of different concentrations such as 10 copies / microliter, 1 μL was added to the RPA reaction system described in Example 1, and the screened primer combination EBO-RPA-F2 / R2 was used to perform RPA detection on the above templates with different copy numbers. Each well was repeated 8 times, and the sensitivity of RPA detection was calculated using Prism software with the molecular copy number and detection time as parameters.

[0061] The result is shown in the figure. Figure 4A It is the real-time amplification curve diagram of different copy numbers, the abscissa is time, and the ordinate is the fluorescence signal value; Figure 4B The semi-logarithmic line graph of the sensitivity of RPA to detect Ebola virus, the abscissa is the copy number, and the ordinate is the threshold time. It can be seen from the figure that the detection...

Embodiment 3

[0062] Embodiment 3: The specificity evaluation that RPA detects

[0063] In the specificity evaluation, influenza virus and adenovirus were used as controls to determine the specificity of the RPA detection method of the present invention.

[0064] The influenza virus RNA and the DNA of the adenovirus were extracted, and RPA detection was carried out respectively, and the adenovirus DNA was detected by RPA according to the method in Example 2; the influenza virus RNA was detected using the following 50 μL RT-RPA system: each 2 μL (10 μmol / L), probe 0.5 μL (10 μmol / L), RNA template 1 μL, RNase inhibitor 0.5 μL, deionized water 12 μL and 29.5 μL buffer into a 0.2 mL TwistAmpRTexo reaction tube containing lyophilized enzyme powder. Then add 2.5 μL of magnesium acetate solution to the lids of all the reaction tubes, mix well and immediately put it into the RPA fluorescence detector for real-time monitoring, and react at 40°C for 20 minutes.

[0065] Results No amplification cur...

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PUM

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Abstract

The invention provides a RPA (recombinase polymerase amplification) kit used for detecting Ebola virus, special-purpose primers and probes of the RPA kit, and applications of the RPA kit in detection of Ebola virus. The RPA kit, the special-purpose primers, and the probes are designed based on Ebola virus NP gene conserved sequence, and the Ebola virus NP gene conserved sequence possesses an oligonucleotide sequence represented by SEQ ID NO.1. It is shown by research results that the RPA kit can be used for rapid detecting of Ebola virus in samples with specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the molecular biology of Ebola virus, in particular to an RPA kit for detecting Ebola virus, special primers and probes thereof and their application in detecting Ebola virus. Background technique [0002] Ebola virus causes an acute infectious disease in humans and primates called Ebola hemorrhagic fever. The incubation period after human infection with Ebola virus is 3-21 days. At the initial stage of the onset, patients present symptoms such as high fever and headache similar to common influenza, and then the disease progresses and deteriorates rapidly, manifesting as hemorrhage, multiple organ failure and shock-like syndrome. Ebola virus belongs to the filoviridae family and contains 5 subtypes, four of which can cause human infection, and the fatality rate can be as high as 90%. In addition, there is currently no effective treatment and vaccine against Ebola in the world. This makes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/70C12Q1/6867
Inventor 陈泽良刘超杨明娟柯跃华汪舟佳黄留玉杜昕颖王雪松
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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