AUTHOR=Xu Jian-Hao , Kang Lin , Yuan Bing , Feng Zi-Han , Li Shi-Qing , Wang Jing , Wang Ya-Ru , Xin Wen-Wen , Gao Shan , Li Jia-Xin , Sun Yan-Song , Wang Jing-Lin , Yuan Yuan 

TITLE=Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.901520

DOI=10.3389/fmicb.2022.901520

ISSN=1664-302X

ABSTRACT=Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, potential to be fatal and aerosol spread, it is regarded as a severe public health threat. The US Centers for Disease Control and Prevention (CDC) have classified it as category A potential agent for bioterrorism and Tier 1 Select Agent. Herein, we combined RPA with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4). creating a two-pronged rapid and ultrasensitive diagnostic methods for detecting F. tularensis. The real-time 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, 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 minute amounts of F. tularensis genomic DNA (2.5 fg). There were no non-specific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, on-site and in epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay for more accurate diagnosis.