Method for detecting bacillus anthracis by combining RPA with CRISPR technology and complete set of reagents

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

Active Publication Date: 2021-03-19
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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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 for detecting bacillus anthracis by combining RPA with CRISPR technology and complete set of reagents
  • Method for detecting bacillus anthracis by combining RPA with CRISPR technology and complete set of reagents
  • Method for detecting bacillus anthracis by combining RPA with CRISPR technology and complete set of reagents

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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 for detecting bacillus anthracis by combining RPA with a CRISPR technology and a complete set of reagents. The invention provides a complete set of primer group for detecting the bacillus anthracis and / or screening bacillus anthracis virulent strains. The complete set of primer group contains three sets of primers specific to chromosome genes BA_5345 and plasmid virulent genes pagA and capA of the bacillus anthracis virulent strains; each set of primer is composed of an RPA primer and crRNA; the RPA primer is designed according to a target gene conserved sequence; the crRNA comprises an anchoring sequence capable of being combined with cas protein and a spacer sequence matched with an RPA primer amplification product sequence. The complete set of primer group has relatively good sensitivity and specificity for sample detection of clinical simulation samples and soil simulation samples, can effectively distinguish the bacillus anthracis virulent strainsfrom other bacteria, provides a rapid detection method for bacillus anthracis in-vitro diagnosis, and has important significance for soil environment screening of the bacillus anthracis in Chinese epidemic areas.

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 Applications(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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