There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) Mycobacterium tuberculosis into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with ∼25 M. tuberculosis genomes (100 fg of M. tuberculosis DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C. The test detected M. tuberculosis in 99.1% of sputum extracts and 100% of sediment extracts and showed 100% concordance with the results of real-time PCR. The levels of concordance between M. tuberculosis and resistance-associated gene detection were 99.1% and 98.4% for sputum and sediment extracts, respectively. Genotyping results were 100% concordant between sputum and sediment extracts. Relative to the results of culture-based drug susceptibility testing, the test was 97.1% specific and 75.0% sensitive for the detection of rifampin resistance in both sputum and sediment extracts. The specificity for the detection of isoniazid (INH) resistance was 98.4% and 96.8% for sputum and sediment extracts, respectively, and the sensitivity for the detection of INH resistance was 63.6%. The amplification microarray reported the correct genotype for all discordant phenotype/genotype results. On the basis of these data, primary sputum may be considered a preferred specimen for the test. The amplification microarray design, shelf life, and analytical performance metrics are well aligned with consensus product profiles for next-generation drug-resistant M. tuberculosis diagnostics and represent a significant ease-of-use advantage over other hybridization-based tests for diagnosing MDR tuberculosis.
The objective of this study was to develop and validate a simple, field-portable, microarray system for monitoring microbial community structure and dynamics in groundwater and subsurface environments, using samples representing site status before acetate injection, during Fe-reduction, in the transition from Fe- to SO(4)(2-)-reduction, and into the SO(4)(2-)-reduction phase. Limits of detection for the array are approximately 10(2)-10(3) cell equivalents of DNA per reaction. Sample-to-answer results for the field deployment were obtained in 4 h. Retrospective analysis of 50 samples showed the expected progression of microbial signatures from Fe- to SO(4)(2-) -reducers with changes in acetate amendment and in situ field conditions. The microarray response for Geobacter was highly correlated with qPCR for the same target gene (R(2) = 0.84). Microarray results were in concordance with quantitative PCR data, aqueous chemistry, site lithology, and the expected microbial community response, indicating that the field-portable microarray is an accurate indicator of microbial presence and response to in situ remediation of a uranium-contaminated site.
bWe developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. M ycobacterium tuberculosis infects one-third of the world's population, with approximately nine million new cases and two million deaths attributable to the disease each year (1). Early case detection and rapid treatment are considered the most effective control strategies to reduce M. tuberculosis transmission (2), especially in cases involving multidrug-resistant (MDR) or extensively drug-resistant (XDR) M. tuberculosis. Culture-based methods remain the gold standard for diagnosing drug-resistant M. tuberculosis but can take several weeks or months to complete. Thus, nucleic acid-based drug susceptibility tests are becoming increasingly attractive as diagnostic tools in order to initiate individualized, patient-appropriate treatment in a timely manner.Technologies such as Cepheid's GeneXpert and Hain line probe assays reduce the time to diagnosis for many tuberculosis (TB) patients, provide a rapid read-out indicating resistance to rifampin or selected mutations conferring resistance to other firstor second-line drugs, and illustrate the potential to deploy molecular tests closer to the point of need (3). However, the number of known genes and mutations conferring resistance to first-and second-line drugs greatly exceeds the multiplexing capacity of these platforms, which may limit their clinical efficacy in the treatment and control of multidrug-resistant or extensively drug-resistant TB. Planar and suspension microarrays are well suited to address the multiple-gene, multiple-mutation challenge of diagnosing drug-resistant TB (4-12), but clinical adoption of microarray technology is hampered by poor reproducibility (13-15), complex workflows, and/or extensive user subjectivity and involvement in image and data analysis (16). In order to translate microarrays into efficacious TB diagnostics at the point of need, it is therefore necessary to simplify user interaction with the technology while retaining the ability to detect multiple genes and multiple mutations in a timely manner. The objectives of this study were to develop a gel element microarray test for MDR TB at a level of coverage surpassing what is currently available with WHO-endorsed molecular platforms, estimate the analytical specificity of the test on M. tuberculosis isolates of known genotype and phenotype, and compare the performance of the test to that of conventional drug susceptibility testing as a precursor to integrating the method into an entirely closed-amplicon consumable (17, 18) and sample-to-answer system. MATERIALS AND METHODSIsolates and positive control...
Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.
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