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Novel DNA Chip Based on a Modified DigiTag2 Assay for High-Throughput Species Identification and Genotyping of Mycobacterium tuberculosis Complex Isolates
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Document Title
Novel DNA Chip Based on a Modified DigiTag2 Assay for High-Throughput Species Identification and Genotyping of Mycobacterium tuberculosis Complex Isolates
Author
Srilohasin P, Chaiprasert A, Tokunaga K, Nao N, Prammananan T
Name from Authors Collection
Affiliations
Mahidol University; University of Tokyo; National Center for Global Health & Medicine - Japan; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
JOURNAL OF CLINICAL MICROBIOLOGY
Year
2014
Volume
52
Issue
6
Page
1962-1968
Open Access
Green Published
Publisher
AMER SOC MICROBIOLOGY
DOI
10.1128/JCM.00153-14
Format
Abstract
A multipurpose high-throughput genotyping tool for the assessment of recent epidemiological data and evolutional pattern in Mycobacterium tuberculosis complex (MTBC) clinical isolates was developed in this study. To facilitate processing, 51 highly informative single nucleotide polymorphisms (SNPs) were selected for discriminating the clinically most relevant MTBC species and genotyping M. tuberculosis into its principle genetic groups (PGGs) and SNP cluster groups (SCGs). Because of the high flexibility of the DigiTag2 assay, the identical protocol and DNA array containing the identical set of probes were applied to the highly GC-rich mycobacterial genome. The specific primers with multiplex amplification and hybridization conditions based on the DigiTag2 principle were optimized and evaluated with 14 MTBC reference strains, 4 nontuberculous mycobacteria (NTM) isolates, and 322 characterized M. tuberculosis clinical isolates. The DNA chip that was developed revealed a 99.85% call rate, a 100% conversion rate, and 99.75% reproducibility. For the accuracy rate, 98.94% of positive calls were consistent with previous molecular characterizations. Our cost-effective technology was capable of simultaneously identifying the MTBC species and the genotypes of 96 M. tuberculosis clinical isolates within 6 h using only simple instruments, such as a thermal cycler, a hybridization oven, and a DNA chip scanner, and less technician skill was required than for other techniques. We demonstrate this approach's potential as a simple, flexible, and rapid tool for providing clearer information regarding circulating MTBC isolates.
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Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
Funding Sponsor
Thailand Research Fund through the Royal Golden Jubilee Ph. D. Program [PHD/0315/2551]; Japan Science and Technology Agency (JST); National Science and Technology Development Agency (NSTDA); Mahidol University Research Fund, a Siriraj Graduate Thesis Scholarship; Drug-Resistant Tuberculosis Research Fund of the Siriraj Foundation; Department of Human Genetics of the Graduate School of Medicine at the University of Tokyo
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