期刊
GENE
卷 857, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.gene.2023.147173
关键词
Drug-resistance; CRISPR-Cas9; Guide RNA; rpoB; RRDR
We developed a Cas9/gRNA-assisted quantitative Real-Time PCR (CARP) assay to detect drug-resistant mutations in Mycobacterium tuberculosis (Mtb). The CARP assay showed high sensitivity and 100% specificity in recognizing single nucleotide changes associated with drug resistance. This low-cost and rapid diagnostic test holds great promise in the diagnosis and prognosis of tuberculosis patients.
A simple, rapid and low-cost diagnostic test, which can detect both the drug-sensitive and the drug-resistant tuberculosis (TB) cases is the need of the hour. Here, we developed a Cas9/gRNA-assisted quantitative Real - Time PCR (qRT-PCR) (CARP) assay to detect single nucleotide mutations causing drug resistance in the TB pathogen, Mycobacterium tuberculosis (Mtb). Guide RNAs (gRNAs) were designed against S531 and H526 posi- tions in the rifampicin (RIF)-resistance-determining region (RRDR) of the Mtb rpoB gene that exhibit frequent mutations in the RR clinical isolates of Mtb. Conditions were optimised for in vitro Cas9 cleavage such that single nucleotide changes at these positions can be recognised by Cas9/gRNA complex with high sensitivity and 100% specificity. Further estimation of Cas9/gRNA-based cleavage of target DNA by qRT-PCR led to rapid detection of drug-resistant sequences. The newly designed CARP assay holds a great deal of promise in the diagnosis and prognosis of patients suffering from TB, in a cost-effective manner.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据