Evaluation of the CombiChip Mycobacteria (TM) Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis
ABSTRACT The CombiChip Mycobacteriatrade mark Drug-Resistance Detection DNA chip, recently developed by GeneIn (Pusan, South Korea), is an oligonucleotide microchip coupled with polymerase chain reaction for the detection of mutations associated with resistance to isoniazid (INH) and rifampin (RIF). This oligonucleotide chip was compared with DNA sequencing and phenotypic drug susceptibility testing with 69 INH- and/or RIF-resistant and 27 all tested drug-susceptible Mycobacterium tuberculosis isolates. Two selected codons (the katG codon 315 and inhA15) allowed identification of 84.1% of INH-resistant isolates and 100% of RIF resistance were detected by screening for 7 codons: rpoB511, rpoB513, rpoB516, rpoB522, rpoB526, rpoB531, and rpoB533. The overall specificity of this oligonucleotide chip for detecting INH and RIF resistance were 100 and 95.3%, respectively. This level of sensitivity and specificity is concordant with that from the determination of M. tuberculosis drug resistance by DNA sequencing. This oligonucleotide chip is a rapid and reliable genotypic method capable of detecting multiple mutations associated with INH and RIF resistance simultaneously in a single microchip slide.
Article: Genotypic Drug Resistance Assays[Show abstract] [Hide abstract]
ABSTRACT: The purpose of this review is to describe the mechanisms of antimicrobial resistance and to present some genotypic drug resistance assays used to detect antimicrobial resistance. Genotypic drug resistance assays that are increasingly used in the clinical microbiology laboratory and their applications in the clinical settings will be further discussed.
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ABSTRACT: Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.Journal of Visualized Experiments 01/2014; DOI:10.3791/51256
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ABSTRACT: To rapidly detect rifampicin (RIF), isoniazid (INH) and multidrug resistance (MDR) in Mycobacterium tuberculosis isolates, a new system (BluePoint MtbDR, Bio Concept Inc., Taichung, Taiwan) including an oligonucleotide array and an automatic reader was evaluated. The array simultaneously identifies M. tuberculosis and predominant mutations in the rpoB, katG and inhA upstream regulatory region (inhA-r) genes. The system was assessed with 324 clinical M. tuberculosis isolates, including 210 MDR, 41 RIF mono-resistant, 34 INH mono-resistant and 39 fully susceptible isolates. The results were compared with those obtained using the GenoType MTBDRplus test, drug-resistant gene sequencing and conventional drug susceptibility testing. The detection limit of the array was 25 pg DNA. The array and the GenoType MTBDRplus test detected 179 (85.2%) and 182 (86.7%) MDR M. tuberculosis strains, respectively. The sensitivities of the array for detecting RIF and INH resistance were 98.4% and 87.7%, respectively, whereas the sensitivities of the GenoType MTBDRplus test for detecting RIF and INH resistance were 98.8% and 88.9%, respectively. No significant difference was found between the tests with respect to their sensitivities to detect MDR (p=0.66), RIF resistance (p=0.69) or INH resistance (p=0.68). The discrepancies were mainly attributed to rare mutations in inhA-r, which were not included in the array. The array can directly reveal transmission-associated mutations, which are useful for epidemiological investigations. The turnaround time of the array test was 6-7 h. This study confirms the feasibility of this system for rapid and accurate diagnosis of INH and RIF resistance in M. tuberculosis. This article is protected by copyright. All rights reserved.Clinical Microbiology and Infection 12/2013; 20(9). DOI:10.1111/1469-0691.12517 · 5.20 Impact Factor