Bacillus anthracis (B. anthracis) is a gram-positive bacterium responsible for the acute disease anthrax. Rapid and reliable identification of pathogenic B. anthracis is important in the detection of natural infectious disease cases or bio-threats. Herein, a DNA endonuclease targeted CRISPR trans reporter (DETECTR) detection platform based on recombinase polymerase amplification (RPA) was studied. The DETECTR system targeted three sequences from B. anthracis (the BA_5345 chromosomal specific marker, the protective antigen gene pag A from pXO1 plasmid and the capsule-biosynthesis-related gene cap A from pXO2 plasmid). We developed a rapid (<40 min), easy-to-implement and accurate identification method for of B. anthracis nucleic acid with near two-copies sensitivity. The combination of tripartite primer sets is effective for the reliable identification of B. anthracis but also for fast screening of pathogenic strains. More importantly, DETECTR correctly detected simulated clinical blood samples and firstly detected positive samples collected from the location of world War-II site, preserved at north-east China (45°36'55.940″ N, 126°38'33.738″ E) with high sensitivity and specificity. Our study provides insight into the DETECTR-based detection of B. anthracis. We present a novel screening and diagnostic option for pathogenic B. anthracis that can be performed on a user-friendly portable device. Based on its proven reliability, sensitivity, specificity and simplicity, our proposed method can be readily adapted to detect pathogenic B. anthracis, anthrax and biothreats.
Keywords: Bacillus anthracis; Biothreats; CRISPR/Cas12a; DETECTR; Isothermal detection; Nucleic acid detection.
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