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CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats-Cas13a) system for detecting coxiella burnetii nucleic acid

A technology of Coxiella basidioides and nucleic acid molecules, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, DNA/RNA fragments, etc., and can solve the problem of complex isolation operation, low efficiency, and ineffective detection of Rickettsia pathogens. Antibody and other issues, to achieve significant application promotion value, rapid and accurate detection effect

Active Publication Date: 2021-01-29
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, serological diagnosis often fails to detect antibodies in the early stages of Q fever
Rickettsia pathogen isolation operation is complex and inefficient

Method used

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  • CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats-Cas13a) system for detecting coxiella burnetii nucleic acid
  • CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats-Cas13a) system for detecting coxiella burnetii nucleic acid
  • CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats-Cas13a) system for detecting coxiella burnetii nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the preparation of each component of kit

[0043] 1. Preparation of LwCas13a protein

[0044] For the expression, purification and activity identification of LwCas13a protein, refer to the method in the patent application document entitled "A crRNA target for detection of Ebola virus and CRISPR-Cas13a system" with publication number CN110628955. Specific steps are as follows:

[0045] 1. Induced expression, purification and identification of LwCas13a protein

[0046]The LwCas13a expression plasmid is PC013-Twinstrep-SUMO-huLwCas13a, obtained from the Addgene platform (https: / / www.addgene.org / search / catalog / plasmids / ?q=PC013-Twinstrep-SUMO-huLwCas13a). The LwCas13a expression plasmid expresses the LwCas13a protein shown in Sequence 8 of the Sequence Listing.

[0047] The LwCas13a expression plasmid was introduced into Escherichia coli Rosetta (DE3) competent cells, and then cultured in TB liquid medium at 37°C and 200rpm for more than 14 hours. Then tra...

Embodiment 2

[0065] Embodiment 2, the screening of optimal crRNA

[0066] The standard plasmid mother liquor prepared in Example 1 is diluted to make the plasmid concentration 10 6 copies / μL is the standard plasmid solution of Plasmid-Coxiella.

[0067] 1. RPA amplification

[0068] Using the Plasmid-Coxiella standard plasmid solution as a template solution, RPA amplification was performed to obtain RPA amplification products.

[0069] The RPA amplification system is shown in Table 1.

[0070] Prepare 47.5 μL of the system shown in Table 1, and add it into the basic reaction unit equipped with RPA freeze-dried powder, so that the freeze-dried powder is fully redissolved and even. Add 2.5 μL of 280 mM magnesium acetate solution to the cap of each reaction tube, close the cap and collect by centrifugation and mix well. Then place the reaction tube at 39° C. for 20-40 minutes to react.

[0071] 2. Detection of Coxella bainiella nucleic acid based on CRISPR-Cas13a system

[0072] The rea...

Embodiment 3

[0077] Embodiment 3, the establishment of the composition of kit and method

[0078] 1. The composition of the kit

[0079] The composition of the kit is as follows: the Cas13a protein prepared in Example 1, the crRNA-1 prepared in Example 1, the standard plasmid stock solution prepared in Example 1, the RPA amplification primers prepared in Example 1 (Coxiella-F and Coxiella-R ).

[0080] Second, the establishment of the method

[0081] 1. RPA amplification

[0082] Using the test DNA solution as the template solution, carry out RPA amplification to obtain the RPA amplification product.

[0083] The test DNA solution is a standard plasmid solution or a sample DNA solution.

[0084] The RPA amplification system is shown in Table 1.

[0085] Table 1 RPA amplification system

[0086]

[0087] Prepare the 47.5 μL system shown in Table 1, and add the basic reaction unit equipped with RPA freeze-dried powder (with nfo RPA amplification kit), the lyophilized powder was fu...

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Abstract

The invention discloses a CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats Cas13a) system for detecting coxiella burnetii nucleic acid. The invention provides crRNA. The crRNAis shown as a sequence 1 in a sequence table. The invention further provides a nucleic acid molecule composition. The nucleic acid molecule composition is composed of a primer Coxiella-F, a primer Coxiella-R and the crRNA shown in the specification. The primer Coxiella-R is a single-stranded DNA (deoxyribonucleic acid) molecule shown as a sequence 7 in the sequence table. The invention also discloses an application of any one of the crRNA or the nucleic acid molecule composition in detection of coxiella burnetii nucleic acid. The invention discloses a CRISPR-Cas13a detection system for detecting coxiella burnetii nucleic acid. The CRISPR-Cas13a detection system achieves higher-sensitivity, high-specificity, rapid and accurate detection on a trace coxiella burnetii nucleic acid sample. Theinvention has important application and popularization value for prevention and control of Coxiella burnetii infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a CRISPR-Cas13a system used for nucleic acid detection of Coxiella béziae. Background technique [0002] Coxiella burnetii is the causative agent of Q fever, an important zoonotic disease. Coxiella beinii is transmitted vertically during the tick menstrual period and via eggs. The ticks carrying Coxiella bezii bite wild animals or domestic animals, causing animal infection; infected animals discharge a large number of Coxiella bezii to pollute the environment, causing human Occurrence or outbreak of Q fever. The natural foci of Q fever are widely distributed in my country. At least 20 provinces, municipalities and autonomous regions have reported the occurrence of Q fever. Among them, Sichuan, Yunnan, Inner Mongolia, Xinjiang and Tibet have also experienced Q fever epidemics. [0003] Serological detection of Coxiella beinii-specific antibodies is currently the gold standard for diag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12Q1/04C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/327C12Q2521/507C12Q2525/161C12Q2563/107Y02A50/30
Inventor 熊小路李浩周冬生孙岩松焦俊欧阳譞付梦姣于永慧赵月峨杨文慧胡凌飞杨慧盈殷喆
Owner ACADEMY OF MILITARY MEDICAL SCI
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