Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform

Seung Heon Lee, Hee Baeg Choi, Sung Yul Yu, Uck Jin Chang, Chang Ki Kim, Hee Jin Kim

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 languageEnglish
Pages (from-to)487-493
Number of pages7
JournalAnnals of Laboratory Medicine
Volume35
Issue number5
DOIs
Publication statusPublished - 2015 Sep 1
Externally publishedYes

Fingerprint

Microspheres
Drug Resistance
Tuberculosis
Alleles
Mutation
Confidence Intervals
Pharmaceutical Preparations
Throughput
Mycobacterium tuberculosis
Sensitivity and Specificity
Nucleic acid sequences
Ethambutol
Costs and Cost Analysis
Isoniazid
Testing
Rifampin
Multiplex Polymerase Chain Reaction
Genetic Predisposition to Disease
Costs
Assays

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 journalArticle

Lee, Seung Heon ; Choi, Hee Baeg ; Yu, Sung Yul ; Chang, Uck Jin ; Kim, Chang Ki ; Kim, Hee Jin. / Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform. In: Annals of Laboratory Medicine. 2015 ; Vol. 35, No. 5. pp. 487-493.
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T1 - Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform

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AU - Choi, Hee Baeg

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AU - Chang, Uck Jin

AU - Kim, Chang Ki

AU - Kim, Hee Jin

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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

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