Abstract
Drug-resistant tuberculosis (TB) is a serious public health problem and threat to global TB prevention and control. Streptomycin (STR) is the earliest and classical anti-TB drug, and it is the earliest drug that generated resistance to anti-TB treatment, which limits its use in treating TB and impedes TB control efforts. The rapid, economical, and highly sensitive detection of STR-resistant TB may help reduce disease transmission and morbimortality. CRISPR/CRISPR-associated protein (Cas) is a new-generation pathogen detection method that can detect single-nucleotide polymorphisms with high sensitivity and good specificity. In this study, a Cas12a RR detection system that can recognize more non-traditional protospacer-adjacent motif-targeting sequences was developed based on Cas12a combined with recombinase polymerase amplification technology. This system detects 0.1% of the target substance, and the entire detection process can be completed within 60 min. Its sensitivity and specificity for detecting clinical STR-resistant Mycobacterium tuberculosis were both 100%. Overall, the Cas12 RR detection system provides a novel alternative for the rapid, simple, sensitive, and specific detection of STR-resistant TB, which may contribute to the prompt treatment and prevention of disease transmission in STR-resistant TB.
Highlights
Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of death globally (Ranganathan et al, 2018; Gupta-Wright et al, 2019; Liu et al, 2020)
The rpsl gene and CRISPR-guided RNA (crRNA) were added to the Cas12a wild-type and Cas12a RR systems, respectively, after having been allowed to react for 15 min at 37◦C
The results showed that RPA can amplify an STR-resistant fragment from 0.05 ng M.tb whole genome with high sensitivity (Figures 5A,D)
Summary
Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of death globally (Ranganathan et al, 2018; Gupta-Wright et al, 2019; Liu et al, 2020). According to the World Health Organization (WHO) 2021 Global TB Report, there were approximately 9.9 million new cases and 1.5 million deaths worldwide in 2020. The irrational use of antibiotics causes drug-resistant TB to become more serious (Wang et al, 2017; Tam et al, 2019; World Health Organization (WHO), 2021). Drug-resistant TB is one of the major reasons for high mortality rates and poses an enormous burden on TB prevention and control (World Health Organization (WHO), 2021). Lys43Arg, and Lys88Arg in the rpsl gene of STR, have mutation rates as high as 70–90%. Detecting the two site mutations will contribute to diagnosing STR resistance (Wang et al, 2019; Islam et al, 2020)
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