Longhorn Vaccines & Diagnostics, San Antonio, Texas, USA.
J Clin Microbiol. 2012 Dec;50(12):3831-7. doi: 10.1128/JCM.01893-12. Epub 2012 Sep 12.
A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes-rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)-were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.
采用 Ion Torrent Personal Genome Machine (PGM) 开发了一种新的全长度结核分枝杆菌一线和二线耐药基因分析方案。扩增并测序了 5 个基因 - rpoB（利福平）、katG（异烟肼）、pncA（吡嗪酰胺）、gyrA（氧氟沙星/氟喹诺酮）和 rrs（氨基糖苷类），并将结果与 2011 年 7 月至 11 月期间在南非收集的 26 个具有地理多样性的临床分离株进行的基因 Hain 线探针分析（LPA）和表型 Bactec MGIT 960 分析的基因型结果进行比较。Ion Torrent 测序与 MGIT 960 培养获得的表型耐药和 LPA 获得的 rpoB 和 katG 基因型结果完全一致（26/26）。在几个利福平耐药的分离株中，Ion Torrent 测序揭示了一些 LPA 没有定义突变的不常见取代（H526R 和 D516G）。重要的是，观察到 rpoB（V194I）、rrs（G878A）和 pncA（Q122Stop）基因中以前未描述的突变。Ion Torrent 测序可能有助于追踪和监测具有地理多样性的耐多药和广泛耐药菌株，并且有可能整合到非洲、印度和中国的选定区域和参考环境中。
