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crRNA target point and CRISPR-Cas13a system for detecting Ebola virus

An Ebola virus and target sequence technology, applied in the field of crRNA target and CRISPR-Cas13a system, can solve the problems of complex sample processing, high instrument requirements, and insufficient sensitivity

Active Publication Date: 2019-12-31
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain deficiencies in the existing detection technologies. The colloidal gold method is simple and portable, but its sensitivity is insufficient; the constant temperature amplification method including LAMP and RPA has high sensitivity, but it is easy to contaminate during operation; the fluorescent quantitative PCR method is stable, Reliable, with a certain sensitivity, but the sample processing is complicated and requires high requirements on the instrument

Method used

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  • crRNA target point and CRISPR-Cas13a system for detecting Ebola virus
  • crRNA target point and CRISPR-Cas13a system for detecting Ebola virus
  • crRNA target point and CRISPR-Cas13a system for detecting Ebola virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, Ebola virus nucleic acid detection kit and detection method based on CRISPR-Cas13a system

[0051] (1) Ebola virus nucleic acid detection kit based on CRISPR-Cas13a system

[0052] 1. LwCas13a protein

[0053] For the expression, purification and activity identification of LwCas13a protein, refer to the method in the patent document with the title of invention "An effective Cas13a-based anti-dengue virus nucleic acid target and its application" and the publication number is CN108715849A. Specific steps are as follows:

[0054] 1. Induced expression, purification and identification of LwCas13a protein

[0055] The LwCas13a expression plasmid Addgene-PC013-Twinstrep-SUMO-huLwCas13a was obtained from the Addgene platform, and the LwCas13a expression plasmid was transferred into Rosetta (DE3) competent cells, cultured in TB liquid medium at 37°C and 200rpm for more than 14 hours, and inserted into new Amp + In the resistant TB medium, culture at 37°C and 300r...

Embodiment 2

[0123] Embodiment 2, the sensitivity detection of the inventive method

[0124] The plasmid standard after serial dilution was used as a template, and the plasmids containing EBOV gene fragments in different concentrations were detected according to the method in Example 1, so as to test the sensitivity of the method of the present invention. Specific steps are as follows:

[0125] 1. Carry out gradient dilution to the plasmid standard substance obtained in Step (1) of Example 1 to obtain plasmid solutions containing different concentrations of EBOV gene fragments: 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL.

[0126] 2. Carry out RAA amplification according to the method in Step (1)-4 of Example 1 to obtain the RAA amplification product.

[0127]3. After being amplified by RAA, take 5 μL of the amplified product to detect Ebola virus nucleic acid based on the CRISPR-Cas13a system according to the metho...

Embodiment 3

[0129] Embodiment 3, the specific detection of the method of the present invention

[0130] Forest encephalitis virus (TBEV), dengue virus type 2 (DENV2), dengue virus type 4 (DENV4), Japanese encephalitis virus (JEV), yellow fever virus (YFV), HBV virus (HBV) and Bennett virus Coxella speii (Cb) pathogenic nucleic acid was used as a template, and different viral nucleic acids were detected according to the method in Example 1, to verify the specificity of the method of the present invention. Specific steps are as follows:

[0131] 1. Ebola virus (EBOV), forest encephalitis virus (TBEV), dengue virus type 2 (DENV2), dengue virus type 4 (DENV4), Japanese encephalitis virus (JEV), yellow fever virus ( YFV), HBV virus (HBV) and Coxella benardii (Cb) nucleic acids were used as detection templates, and RAA amplification was performed according to the method in step (1) of Example 1, to obtain RAA amplification products.

[0132] 2. After being amplified by RAA, take 5 μL of the a...

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Abstract

The invention discloses a crRNA target point and CRISPR-Cas13a system for detecting an Ebola virus. The CRISPR-Cas13a system comprises Cas13a protein and crRNA, or a composite formed from the Cas13a protein and the crRNA, and the crRNA comprises an anchoring sequence being combined with the Cas13a protein and a guiding sequence targeted to the sequence of the target point of the Ebola virus; and the sequence of the target point of the Ebola virus is located at the 1001-1028th of the genome of the Ebola virus. The crRNA can realize high-sensitivity and high-specifity detection on the nucleic acid of the Ebola virus by activating Cas13a, and the sensitivity achieves 1 copy / test (1copy / test).

Description

technical field [0001] The invention relates to a crRNA target for detecting Ebola virus and a CRISPR-Cas13a system, belonging to the technical field of molecular diagnosis. Background technique [0002] Ebola hemorrhagic fever is a severe infectious disease caused by Ebola virus (EBOV) that causes acute infection in humans and non-human primates. The characteristics of specific drugs and no vaccine prevention are mainly prevalent in Central Africa and West Africa, and its mortality rate is as high as 50% to 90%. At present, the Ebola epidemic has constituted a public health emergency of international concern. It is considered to be one of the most ferocious diseases in the world, and it is also a major problem facing global public health. Ebola virus detection techniques include virus isolation and culture, detection of IgM and IgG antibodies, fluorescent quantitative PCR, constant temperature nucleic acid detection technology, immunohistochemical technology, etc. However...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/113C12N9/22
CPCC12Q1/701C12Q1/686C12N15/113C12N9/22C12N2310/20C12Q2563/107Y02A50/30
Inventor 李浩孙岩松董雪王彦贺寇志华周育森杨明娟
Owner ACADEMY OF MILITARY MEDICAL SCI
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