Singh S P, Salamon H, Lahti C J, Farid-Moyer M, Small P M
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, California 94305, USA.
J Clin Microbiol. 1999 Jun;37(6):1927-31. doi: 10.1128/JCM.37.6.1927-1931.1999.
Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which has provided important insights into the epidemiology and population biology of many pathogens. However, few studies have used PFGE for the molecular epidemiology of Mycobacterium tuberculosis. A laboratory protocol was developed to determine the typeability, stability, and reproducibility of PFGE typing of M. tuberculosis. Formal data-analytical techniques were used to assess the genetic diversity elucidated by PFGE analyses using four separate restriction enzymes and by IS6110 RFLP analyses, as well as to assess the concordance among these typing methods. One hundred epidemiologically characterized clinical isolates of M. tuberculosis were genotyped with four different PFGE enzymes (AseI, DraI, SpeI, and XbaI), as well as by RFLP analysis with IS6110. Identical patterns were found among 34 isolates known to be genetically related, suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates representing population-sampled cases, heterozygosity and information content dependency estimates indicate that all five genotyping systems capture quantitatively similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses and IS6110 typing reveals that PFGE provided more discrimination among isolates with fewer than five copies of IS6110 and less clustering in isolates with five or more copies. The comparisons confirm the hypothesis that the resolution of IS6110 RFLP genotyping is dependent upon the number of IS6110 elements in the genome of isolates. The general concordance among the results obtained with four independent enzymes suggests that M. tuberculosis is a clonal organism. The availability of a robust genotyping technique largely independent of repetitive elements has implications for the molecular epidemiology of M. tuberculosis.
脉冲场凝胶电泳（PFGE）是一种强大的分子生物学技术，它为许多病原体的流行病学和群体生物学提供了重要见解。然而，很少有研究将PFGE用于结核分枝杆菌的分子流行病学研究。我们制定了一项实验室方案，以确定结核分枝杆菌PFGE分型的可分型性、稳定性和可重复性。使用正式的数据分析技术来评估通过四种不同的限制性内切酶进行PFGE分析以及通过IS6110限制性片段长度多态性（RFLP）分析所阐明的遗传多样性，并评估这些分型方法之间的一致性。对100株经过流行病学特征分析的结核分枝杆菌临床分离株进行基因分型，使用四种不同的PFGE酶（AseI、DraI、SpeI和XbaI），以及通过IS6110进行RFLP分析。在已知具有遗传相关性的34株分离株中发现了相同的图谱，这表明PFGE方案是可靠且可重复的。在代表群体抽样病例的66株分离株中，杂合性和信息含量依赖性估计表明，所有五种基因分型系统捕获的遗传多样性水平在数量上相似。然而，PFGE分析与IS6110分型之间的比较表明，PFGE在IS6110拷贝数少于五个的分离株中提供了更多的区分度，而在IS6110拷贝数为五个或更多的分离株中聚类较少。这些比较证实了以下假设：IS6110 RFLP基因分型的分辨率取决于分离株基因组中IS6110元件的数量。使用四种独立酶获得的结果之间的总体一致性表明结核分枝杆菌是一种克隆性生物体。一种基本独立于重复元件的可靠基因分型技术的可用性对结核分枝杆菌的分子流行病学具有重要意义。
