Visual Detection ofrpoBMutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

Abstract

dMultidrug-resistant Mycobacterium tuberculosis is resistant to two first-line antituberculosis drugs, isoniazid and rifampin, resulting in the relapse of tuberculosis. M. tuberculosis grows very slowly, and thus traditional examination methods take time to test its drug resistance and cannot meet clinical needs. The use of a DNA probe makes it possible to test rifampin resistance. We developed an asymmetrical split-assembly DNA peroxidase assay to detect drug-resistant mutation of rifampin-resistant M. tuberculosis in the rpoB gene rapidly and visibly. A new strategy was also designed to eliminate the adverse effects caused by the complicated secondary structure of the target DNA and to improve the efficiency of the probes. This detection system consists of five group detections, covers rifampin-resistant determination region of the rpoB gene, and tests 40 kinds of mutations, including the most common mutations at codons 531 and 526. Every group detection or individual mutant allele detection can distinguish corresponding mutant DNA sequences from the wild-type DNA sequences.

Redefining MTBDR<I>plus</I> test results: what do indeterminate results actually mean?

“…The occurrence of NI results where the wild type probes failed to hybridize to their corresponding sequences could result from the inaccessibility of the genomic sequence in a given sample, such as when secondary DNA structures are present. This problem has been noted in rpoB hybridization, in particular, and might play a role in the observed high occurrence (27.5%) of NI results proven RIF-susceptible in this study [36]. …”

MTB-DR-RIF 9G test: Detection and discrimination of tuberculosis and multi-drug resistant tuberculosis strains

“…S1). It is reported that after asymmetric PCR, a Cy5 labeled ssDNAs in the PCR product of the MTB undergoes self-dimerization due to the complementary base pairs in its sequence [27]. Therefore, a splitter DNA was used to separate the strands of the Cy5 labeled ssDNA in the dimerized form.…”

G-Quadruplexes as An Alternative Recognition Element in Disease-Related Target Sensing

“…Hence, a number of studies resorted to splitting the unabridged G-rich strand into an asymmetric ratio instead [63,64]. In a system involving the asymmetrical split DNAzyme strategy to rapidly and visibly detect rifampin-resistant M.tb with mutations in the rpoB gene, unequal ratio (3:1) of probe A and probe B were assembled through hybridization with the target ssDNA [64]. Since the target DNA was originally in dsDNA form, asymmetric PCR was applied to produce ssDNA.…”

“…The formed DNA G4 is then converted into a DNAzyme that will subsequently catalyze the H 2 O 2 -mediated oxidation of ABTS 2− through peroxidation-like activity when bound with hemin. As a result, an oxidation product, ABTS − was formed and a visible green color was observed [64].…”