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.
| Original language | English |
|---|---|
| Pages (from-to) | 487-493 |
| Number of pages | 7 |
| Journal | Annals of Laboratory Medicine |
| Volume | 35 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 2015 Sep 1 |
| Externally published | Yes |
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Keywords
- Allele-specific primer extension
- Drug-resistance
- Genotyping
- Mycobacteirum tuberculosis
ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, medical
Cite this
Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform. / Lee, Seung Heon; Choi, Hee Baeg; Yu, Sung Yul; Chang, Uck Jin; Kim, Chang Ki; Kim, Hee Jin.
In: Annals of Laboratory Medicine, Vol. 35, No. 5, 01.09.2015, p. 487-493.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform
AU - Lee, Seung Heon
AU - Choi, Hee Baeg
AU - Yu, Sung Yul
AU - Chang, Uck Jin
AU - Kim, Chang Ki
AU - Kim, Hee Jin
PY - 2015/9/1
Y1 - 2015/9/1
N2 - 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.
AB - 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.
KW - Allele-specific primer extension
KW - Drug-resistance
KW - Genotyping
KW - Mycobacteirum tuberculosis
UR - http://www.scopus.com/inward/record.url?scp=84942253328&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84942253328&partnerID=8YFLogxK
U2 - 10.3343/alm.2015.35.5.487
DO - 10.3343/alm.2015.35.5.487
M3 - Article
C2 - 26206684
AN - SCOPUS:84942253328
VL - 35
SP - 487
EP - 493
JO - Annals of Laboratory Medicine
JF - Annals of Laboratory Medicine
SN - 2234-3806
IS - 5
ER -