Kim Kyungjong, Yang Jeong Seong, Choi Hee Baeg, Lee Seung Heon
Korean Institute of Tuberculosis, 168-5, Osongsaegmyeong 4-ro, Osong-eup, Heungduk-gu, Cheongju 28158, Republic of Korea.
Genes Laboratories, 388, Dunchondaero, Jungwon-gu, Seongnam 13403, Republic of Korea.
J Microbiol Methods. 2018 Jan;144:111-116. doi: 10.1016/j.mimet.2017.11.007. Epub 2017 Nov 9.
Molecular drug susceptibility testing (DST) for antituberculosis drugs is important for improving the efficacy of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) treatment. In this study, we developed a molecular high-throughput assay system based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres, referred to here as TAG-ASPE, which can detect mutations related to resistance to injectable second-line drugs and fluoroquinolones. Target genes were amplified by multiplex PCR using DNA from H37Rv and 190 clinical Mycobacterium tuberculosis strains and extended by ASPE using 22 ASPE primers. ASPE products were then sorted on the TAG-ASPE array and detected using a Luminex 200 system. The performance of the TAG-ASPE method was compared with that of sequencing and phenotypic DST. Comparison of the TAG-ASPE method with sequencing showed that the sensitivity and specificity of the TAG-ASPE method were 100% [95% confidence interval (CI), 96.38-100%] and 100% (95% CI, 95.70-100%) for the rrs gene and 100% (95% CI, 96.90-100%) and 100% (95% CI, 95.07-100%) for the gyrA gene, respectively. Compared with phenotypic DST, the sensitivity and specificity of the TAG-ASPE method for detecting drug-resistance mutations against injectable second-line drugs were 92.52% (95% CI, 85.8-96.72%) and 98.7% (95% CI, 92.98-99.97%), respectively. Additionally, the sensitivity and specificity for fluoroquinolone-resistance detection were 85.4% (95% CI, 78.36-90.85%) and 100% (95% CI, 92.38-100%), respectively. The results of this study demonstrate that the TAG-ASPE method can effectively detect mutations conferring resistance to second-line antituberculosis drugs in numerous clinical specimens.
抗结核药物的分子药敏试验（DST）对于提高耐多药结核病（MDR-TB）和广泛耐药结核病（XDR-TB）治疗的疗效至关重要。在本研究中，我们开发了一种基于等位基因特异性引物延伸（ASPE）和MagPlex-TAG微球的分子高通量检测系统，在此称为TAG-ASPE，它可以检测与对注射用二线药物和氟喹诺酮类药物耐药相关的突变。使用来自H37Rv和190株临床结核分枝杆菌菌株的DNA通过多重PCR扩增靶基因，并使用22条ASPE引物通过ASPE进行延伸。然后将ASPE产物在TAG-ASPE阵列上进行分选，并使用Luminex 200系统进行检测。将TAG-ASPE方法的性能与测序和表型DST的性能进行比较。TAG-ASPE方法与测序的比较表明，对于rrs基因，TAG-ASPE方法的灵敏度和特异性分别为100%[95%置信区间（CI），96.38 - 100%]和100%（95%CI，95.70 - 100%），对于gyrA基因分别为100%（95%CI，96.90 - 100%）和100%（95%CI，95.07 - 100%）。与表型DST相比，TAG-ASPE方法检测对注射用二线药物耐药突变的灵敏度和特异性分别为92.52%（95%CI，85.8 - 96.72%）和98.7%（95%CI，92.98 - 99.97%）。此外，氟喹诺酮耐药检测的灵敏度和特异性分别为85.4%（95%CI，78.36 - 90.85%）和100%（95%CI，92.38 - 100%）。本研究结果表明，TAG-ASPE方法可以有效地检测大量临床标本中赋予对二线抗结核药物耐药性的突变。
