A Highly Sensitive and Specific Detection Method for Mycobacterium tuberculosis Fluoroquinolone Resistance Mutations Utilizing the CRISPR-Cas13a System

Frontiers in Microbiology

Tiwei Fu¹  Yuliang Liu²  Keli Qian²  Quanxin Long³  Haijun Deng³  Panqi Gao³  Ailong Huang³  Xiaoxia Cao³  Huizhi Zheng³  Xiaopeng Bai³  Miaomiao Han³  Yuan Hu³  Yaoqin Lu⁴  Jiandong Yang⁴ 

[1] Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing Medical University Stomatology College, Chongqing, China;Department of Infection Control, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China;Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China;Urumqi Municipal Centre for Disease Control and Prevention, Xinjiang, China;

关键词: CRISPR-cas13a;    crRNA screening;    Mycobacterium tuberculosis;    fluoroquinolone resistance mutations;    single-base resolution DNA detection;   

DOI  :  10.3389/fmicb.2022.847373

来源: DOAJ

【 摘 要 】

ObjectivesCRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear.MethodsA combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed.ResultsA set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated Mycobacterium tuberculosis strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated M. tuberculosis, the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays.ConclusionThese results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.

【 授权许可】

Unknown