RPB0237

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Mycobacterium tuberculosis Mycobacterium tuberculosis, Bacterium tuberculosis, Bacillus tuberculosis, Mycobacterium tuberculosis variant tuberculosis 1773 Corynebacteriales Mycobacteriaceae Mycobacterium Mycobacterium tuberculosis Bacterium

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
RAA-F AATTCTAATACGACTCACTATAGGAGATGCACCGTCGAACGGCTGATGACCAAAC 55 0.4 µM 45.45 69.17 16905.05 \
RAA-R CCAGGTCGACACATAGGTGAGGTCTGCTACCCACA 35 0.4 µM 57.14 69.83 10726.01 \

Gene Description

Target Gene GenBank ID
IS6110 genes \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool RAA-CRISPR-MTB \ 30 min 39 °C CRISPR-Cas13a System 1 copies 0.69 100 %

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2024 Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System Qiao Li,Nenhan Wang,Mengdi Pang,Honghao Miao,Xiaowei Dai,Bo Li,Xinyu Yang,Chuanyou Li,Yi Liu Microorganisms 39203350 10.3390/microorganisms12081507

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System

Author(s):

Qiao Li,Nenhan Wang,Mengdi Pang,Honghao Miao,Xiaowei Dai,Bo Li,Xinyu Yang,Chuanyou Li,Yi Liu

Journal:

Microorganisms

Year:

2024

Abstract:

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.