Identification of Rifampin-Resistant Mycobacterium tuberculosis Strains by Hybridization, PCR, and Ligase Detection Reaction on Oligonucleotide Microchips

Abstract

Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches-hybridization, PCR, and ligase detection reaction-were designed to analyze an 81-bp fragment of the gene rpoB encoding the ␤-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

Microfluidic Device for Rapid (<15 min) Automated Microarray Hybridization

“…Microarrays allow several thousands of captured nucleic acid probes to be spotted on a small surface on a solid support, generally a glass slide (1)(2)(3)(4). Efforts have been undertaken to adapt the microarray technology for rapid identification of biomolecules by means of signal transduction after binding to specific probes attached to a solid support (5)(6)(7)(8)(9)(10). Per se, there is a need for a rapid (less than 1 h) and sensitive microarray system suitable for the molecular diagnosis of infectious diseases, which involves the detection of a wide variety of microbial pathogens as well as associated virulence genes such as antimicrobial resistance genes or toxin genes (11,12 ).…”

DNA microarrays and their applications in medical microbiology

“…Oligonucleotide based DNA arrays have been used for parallel species identification and rifampin resistancerelated mutations in mycobacteria (Troesch et al, 1999) and, more specifically, for the detection of M. tuberculosis strains that are resistant to rifampin (Yue et al, 2004) or isoniazid, kanamycin, streptomycin, pyrazinamide, and ethambutol (Gegia et al, 2008). Oligonucleotide microarrays were developed to analyze and identify drugresistant M. tuberculosis strains, and it was found that the results were comparable with those of standard antimicrobial susceptibility testing (Strizhkov et al, 2000;Mikhailovich et al, 2001). A low-cost and -density DNA microarray was designed to detect mutations that confer isoniazid and rifampin resistance in M. tuberculosis isolates.…”

Pulmonary Tuberculosis and Mycobacterium Tuberculosis: Modern Molecular Epidemiology and Perspectives

“…Jalava and Marttila [91] described that high-density oligonucleotide arrays may also offer a powerful solution to genotypic detection of drug-resistant MTB isolates. So far, these microarrays have mainly been used for the detection of rifampin resistance [72,123,198] with promising results. Consequently, Jalava and Marttila [91] argue that the DNA microarray strategy could be expanded to include parallel testing of various genes mediating drug resistance in MTB.…”

“…This method can discriminate single-base differences. On-chip ligase detection reaction is applied to identify approximately 1% of mutant sequences in model samples consisting of mixtures of DNA from wild-type and resistant strains (see [123] for details).These technologies may be useful for clinical research in developed countries, but remain inaccessible for a TB control program on a large scale including low-resource countries.…”

“…First, the technique should have a high sensitivity because the amount of MTB cells in sputum varies and can be very low. Secondly, it should be able to detect minor drug-resistant subpopulations in a sample when the majority of the bacilli are susceptible.Two methods could help in this challenge: the invader assay [32] and on-chip ligase detection reaction [123]. The invader assay uses the thermostable flap endonuclease Cleavase VIII, derived from Following purification and quality control, aliquots (~5 nl) are printed on coated glass microscope slides using a computer-controlled, high-speed robot.Total RNA from both the test and reference sample is fluorescently labeled using a single round of reverse transcription.…”