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KMID : 1100720150350050487
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Annals of Laboratory Medicine
2015 Volume.35 No. 5 p.487 ~ p.493
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Detection of First-Line Anti-Tuberculosis Drug Resistance Mutations by Allele-Specific Primer Extension on a Microsphere-Based Platform
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Lee Seung-Heon
Choi Hee-Baeg
Yu Sung-Yul
Chang Uck-Jin
Kim Chang-Ki
Kim Hee-Jin
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Abstract
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Background: Resistance of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs is almost exclusively due to spontaneous chromosomal mutations in target genes. Rapid detection of drug resistance to both first- and second-line anti-TB drugs has become a key component of TB control programs. Technologies that allow rapid, cost-effective, and high-throughput detection of specific nucleic acid sequences are needed. This study was to develop a high-throughput assay based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres to detect anti-TB drug resistance mutations.
Methods: DNA samples from 357 M. tuberculosis clinical isolates and H37Rv were amplified by multiplex PCR using four primer sets, followed by multiplex ASPE using 23 TAG-ASPE primers. The products were sorted on the TAG-ASPE array and detected by using the Luminex xMAP system. Genotypes were also determined by sequencing.
Results: Genetic drug susceptibility typing by the TAG-ASPE method was 100% concordant with those obtained by sequencing. Compared with phenotypic drug susceptibility testing (DST) as a reference method, the sensitivity and specificity of the TAG-ASPE method were 83% (95% confidence interval [CI], 79-88%) and 97% (95% CI, 90-100%) for isoniazid. For rifampin testing, the sensitivity and specificity were 90% (95% CI, 86-93%) and 100% (95% CI, 99-100%). Also, the sensitivity and specificity were 58% (95% CI, 51-65%) and 86% (95% CI, 79-93%) for ethambutol.
Conclusions: This study demonstrated the TAG-ASPE method is suitable for highly reproducible, cost-effective, and high-throughput clinical genotyping applications.
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KEYWORD
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Mycobacteirum tuberculosis, Allele-specific primer extension, Genotyping, Drug-resistance
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