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Method and complete set of reagents for detection of Bacillus anthracis by RPA combined with CRISPR technology

A technology of Bacillus anthracis and Bacillus anthracis, applied in the biological field, can solve the problem of inability to improve sensitivity

Active Publication Date: 2021-07-16
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
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Problems solved by technology

However, in principle, the essence of its single-stage amplification reaction has not changed. Compared with most current real-time quantitative PCR (q-PCR) detection methods, there is no substantial improvement in sensitivity.

Method used

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  • Method and complete set of reagents for detection of Bacillus anthracis by RPA combined with CRISPR technology
  • Method and complete set of reagents for detection of Bacillus anthracis by RPA combined with CRISPR technology
  • Method and complete set of reagents for detection of Bacillus anthracis by RPA combined with CRISPR technology

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Embodiment 1

[0065] Example 1. Design and screening of CRISPR-Cas12a primers for screening virulent strains of Bacillus anthracis

[0066] 1. Design sequence

[0067] 1. Select the target sequence

[0068] On the basis of previous studies, the inventor selected the conserved sequence of the chromosome-specific sequence BA_5345 of Bacillus anthracis (as shown in SEQ ID No.1) and the conserved sequence of the plasmid virulence gene pagA (as shown in Shown in SEQ ID No.2) and the conserved sequence of the plasmid virulence gene capA (shown in SEQ ID No.3) are the target sequences, and the matching of the three sequences can specifically detect the virulent strain of Bacillus anthracis.

[0069] 2. Design of amplification primer pair and crRNA

[0070] Aiming at the above-mentioned chromosome-specific gene BA_5345 of Bacillus anthracis, and the specific conserved sequences of plasmid virulence genes pagA and capA, multiple RPA amplification primer pairs were designed: Rpa-5345-F1 / Rpa-5345-R1...

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Abstract

The invention discloses a method and a complete set of reagents for detecting Bacillus anthracis with RPA combined with CRISPR technology. The present invention provides a set of primers for detecting Bacillus anthracis and / or screening virulent strains of Bacillus anthracis, including three sets of primers specific to the chromosomal gene BA_5345 of the virulent strain of Bacillus anthracis, the plasmid virulence genes pagA and capA, Each set of primers is composed of RPA primers and crRNA, and the RPA primers are designed according to the conserved sequence of the target gene, and the crRNA includes an anchor sequence capable of binding to the cas protein and a spacer sequence matching the sequence of the amplification product of the RPA primer. The complete primer set of the present invention has good sensitivity and specificity for detection of clinical simulation samples and soil simulation samples, can effectively distinguish virulent strains of Bacillus anthracis from other bacteria, and provides a rapid detection method for in vitro diagnosis of Bacillus anthracis. Soil environmental screening of Bacillus anthracis in epidemic areas in China is of great significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a complete set of reagents for detecting Bacillus anthracis with RPA combined with CRISPR technology. Background technique [0002] Bacillus anthracis (Bacillus anthracis) is the first pathogenic bacterium discovered in human history, and it is also an important zoonotic pathogen that causes anthrax natural foci and severe zoonotic infectious diseases. Yunnan, Guizhou, Xinjiang and other places in the agricultural and animal husbandry areas of western my country are high-incidence areas of human anthrax. In addition, because anthrax spores are extremely resistant in the natural environment and easy to produce, they have always been listed as the number one biological warfare agent in the military. In addition to the chromosomal genome, Bacillus anthracis contains two virulence plasmids pXO1 and pXO2 which are closely related to its virulence. Combined detection of spec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/07
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101C12Q2521/327
Inventor 袁媛王景林孙岩松王菁辛文文康琳李岩伟
Owner ACADEMY OF MILITARY MEDICAL SCI
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