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Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis
- Source :
- Frontiers in Microbiology, Vol 13 (2022)
- Publication Year :
- 2022
- Publisher :
- Frontiers Media S.A., 2022.
-
Abstract
- Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis.
Details
- Language :
- English
- ISSN :
- 1664302X
- Volume :
- 13
- Database :
- Directory of Open Access Journals
- Journal :
- Frontiers in Microbiology
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.5f3cb406d690416aa494b2b9e560754e
- Document Type :
- article
- Full Text :
- https://doi.org/10.3389/fmicb.2022.901520