Cascade of deoxyribozymes for the colorimetric analysis of drug resistance in Mycobacterium tuberculosis

Abstract

A visual cascade detection system has been applied to the detection and analysis of drugresistance profile of Mycobacterium tuberculosis complex (MTC), a causative agent of tuberculosis. The cascade system utilizes highly selective split RNA-cleaving deoxyribozyme (sDz) sensors. When activated by a complementary nucleic acid, sDz releases the peroxidase-like deoxyribozyme apoenzyme, which, in complex with a hemin cofactor, catalyzes the color changes of the sample's solution. The excellent selectivity of the cascade has allowed for the detection of point mutations in the sequences of the MTC rpoB, katG, and gyrA genes, which are responsible for resistance to rifampin, isoniazid, and fluoroquinolone, respectively. When combined with isothermal nucleic acid sequence based amplification (NASBA), the assay was able to detect amplicons of 16S rRNA and katG mRNA generated from 0.1 pg and 10 pg total RNA taken for NASBA, respectively, in less than 2 h, producing a signal detectable with the naked eye. The proposed assay may become a prototype for point-of-care diagnosis of drug resistant bacteria with visual signal output.

Diagnostic assay and technology advancement for detecting SARS-CoV-2 infections causing the COVID-19 pandemic

“…Isothermal amplification methods use a single temperature and do not require thermal cycling. These techniques include LAMP [67], recombinase polymerase amplification (RPA) [68], transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA) [69,70], and rolling circle amplification (RCA) [71] and have been used for pathogens detection. The RT-NASBA assay was developed for detection of SARS-CoV [72] and the sensitivity was equivalent to real-time RT-PCR assay.…”

Current and Future Technologies for the Detection of Antibiotic-Resistant Bacteria

“…Dhar et al [229] devised a biosensor that is capable of concurrently detecting point mutations in the rpbB, katG, and gyrA genes of MDR Mycobacterium tuberculosis. This is achieved through the utilization of split deoxyribozyme cascade probes.…”

DNAzyme Nanomachine with Fluorogenic Substrate Delivery Function: Advancing Sensitivity in Nucleic Acid Detection

“…Furthermore, the amplification can also lead to some false positive results due to sample degradation or contamination . Hence, developing an amplification-free detection system would solve the problem of using equipment and consumables that require specialized laboratories and/or qualified personnel to address. , Laboratories could take advantage of visual − or tactile , signal outputs for user-friendly and equipment-free detection formats. However, such assays demonstrate a high limit of detection (LOD) and therefore, remain uncompetitive with nucleic acid amplification-based detection technologies …”