Rapid and Ultrasensitive Approach for the Simultaneous Detection of Multilocus Mutations to Distinguish Rifampicin-Resistant Mycobacterium tuberculosis

Abstract

The untested empirical medications exacerbated the development of multidrug-resistant Mycobacterium tuberculosis (MDR-TB). Here, we develop a rapid and specific method based on loop-mediated isothermal amplification and duplex-specific nuclease for distinguishing rifampicin-resistant M. tuberculosis. Three probes were designed for the codons 516, 526, and 531 on the RNA polymerase β-subunit (rpoB) gene. These three sites accounted for more than 90% of the total mutations of the ropB gene in the rifampicin-resistant strain. The approach can perform simultaneous and sensitive detection of three mutant sites with the actual detection limit as 10 aM of DNA and 62.5 cfu·mL–1 of bacteria in 67 min under isothermal conditions. Moreover, the positive mode of the approach for MDR-TB can not only deal with the randomness and diversity of mutations but also provide an easier way for medical staff to read the results. Therefore, it is a particularly valuable method to handle major and urgent MDR-TB diagnostics.

OWL2: a molecular beacon-based nanostructure for highly selective detection of single-nucleotide variations in folded nucleic acids

“…[1][2][3] The identification of specific SNVs aids in the management of human genetic disorders, and early SNV detection in clinically relevant microbes is crucial in treating infections caused by drug resistant pathogens. [4][5][6][7][8] Traditional methods for SNV detection include DNA sequencing, polymerase chain reaction (PCR) with melting curve analysis, 9 and hybridization assays. DNA sequencing, such as next-generation sequencing (NGS), requires expensive instrumentation and a significant amount of time for data processing.…”

Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays

“…Some drugs can treat it with good results, such as the first-line drugs including isoniazid (INH) and rifampicin (RIF) and the second-line drugs including moxifloxacin (MXF), kanamycin (KAN), capreomycin (CAP), and ethionamide (ETH). , However, the empiric treatment and the widespread use of the drugs without diagnosis lead to the development of drug resistance, for example, multidrug-resistant (MDR), extensively drug-resistant (XDR), extremely drug-resistant (XXDR), and totally drug-resistant (TDR) strains. − This poses a huge challenge for the complete eradication of TB. WHO has set the goal of ending the TB epidemic by 2035 and eradicating TB by 2050 . Therefore, it is particularly important to distinguish between drug-resistant and wild-type strains for the right medicine.…”

“…WHO has set the goal of ending the TB epidemic by 2035 and eradicating TB by 2050. 8 Therefore, it is particularly important to distinguish between drug-resistant and wild-type strains for the right medicine. TB drug susceptibility testing includes solid or liquid culture, 9 sequencing, 10 and realtime quantitative PCR (qPCR).…”