Detection of First-Line Anti-Tuberculosis Drug Resistance Mutations by Allele-Specific Primer Extension on a Microsphere-Based Platform.
10.3343/alm.2015.35.5.487
- Author:
Seung Heon LEE
1
;
Hee Baeg CHOI
;
Sung Yul YU
;
Uck Jin CHANG
;
Chang Ki KIM
;
Hee Jin KIM
Author Information
1. Korean Institute of Tuberculosis, Cheongju, Korea. seung6992@hanmail.net
- Publication Type:Original Article
- Keywords:
Mycobacteirum tuberculosis;
Allele-specific primer extension;
Genotyping;
Drug-resistance
- MeSH:
DNA;
Drug Resistance*;
Ethambutol;
Genotype;
Isoniazid;
Microspheres;
Multiplex Polymerase Chain Reaction;
Mycobacterium tuberculosis;
Rifampin;
Tuberculosis;
Viperidae
- From:Annals of Laboratory Medicine
2015;35(5):487-493
- CountryRepublic of Korea
- Language:English
-
Abstract:
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.
