Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.
Keyphrases
- mycobacterium tuberculosis
- crispr cas
- genome editing
- pulmonary tuberculosis
- loop mediated isothermal amplification
- genome wide
- label free
- high throughput
- public health
- molecularly imprinted
- real time pcr
- escherichia coli
- dna methylation
- emergency department
- copy number
- gene expression
- nucleic acid
- mass spectrometry
- human immunodeficiency virus
- single molecule
- high resolution
- transcription factor
- liquid chromatography
- single cell
- antiretroviral therapy
- adverse drug