Mycobacteria/Nocardia Laboratory, The University of Texas Health Science Center at Tyler, Tyler, Texas, USA.
J Clin Microbiol. 2013 Feb;51(2):409-16. doi: 10.1128/JCM.02443-12. Epub 2012 Nov 21.
Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the "gold standard" of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.
菌株比较对于群体遗传学和评估鸟分枝杆菌复合群（MAC）肺部疾病患者的复发非常重要，但脉冲场凝胶电泳（PFGE）的“金标准”既耗时又复杂。我们使用可变数目串联重复（VNTR）对支气管扩张症患者的细胞内分枝杆菌呼吸道分离株进行指纹图谱分析，建立了一个基于非序列的群体分析数据库。通过 16S rRNA 基因多重 PCR 对 PFGE 鉴定的不同基因型进行物种鉴定。通过内部转录间隔区 1（ITS1）测序确认细胞内分枝杆菌的基因型，并使用 7 个 VNTR 引物进行特征描述。VNTR 扩增子大小和重复数的模式定义了每个特定的 VNTR 类型。在 84 种基因型中鉴定出 42 种 VNTR 类型。PFGE 显示大多数具有相同 VNTR 类型的分离株为克隆或表现出类似的带分组。与 PFGE 相比，重复序列基 PCR（rep-PCR）显示 VNTR 类型之间的模式多样性最小。使用 VNTR 结合 16S 多重 PCR 对 31 名治疗患者的复发分离株进行指纹图谱分析，能够明确可靠地区分来自同一患者的不同基因型，结果与 PFGE 相当。VNTR 进行菌株比较比 PFGE 更容易、更快，与 PFGE 一样准确，并且不需要测序。从 167 株细胞内分枝杆菌分离株开始，VNTR 将细胞内分枝杆菌分为 42 个克隆群。现在可以比较来自不同地理区域、栖息地和临床环境的分离株。
