Center for Global Health, Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical College, New York, New York, USA.
J Clin Microbiol. 2013 Jul;51(7):2232-7. doi: 10.1128/JCM.00268-13. Epub 2013 May 8.
Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct "spacers" separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.
结核分枝杆菌菌株的基因分型对于理解结核病传播动力学和设计疾病防控措施至关重要。不幸的是，这种分型方法涉及复杂的实验室分析，费用昂贵，且需要高水平的技术专长，这使其在资源匮乏国家的应用受到限制，而这些国家正是大多数结核病病例发生的地方。 spoligotyping 是一种基于 PCR 的结核分枝杆菌复合群基因分型方法，具有技术简单、数值输出和高重现性等优点。它基于结核分枝杆菌基因组直接重复区中插入元件之间存在或不存在 43 个不同“间隔物”。 spoligotyping 检测法涉及将 PCR 产物反向杂交到固定在硝酸纤维素膜或微球上的捕获间隔物上。在此，我们报告了使用新一代 Luminex 多重化技术对经典的 43 间隔物方法进行了修改，该技术使用磁性微球。该方法在我们在海地的实验室中对具有已知 spoligotype 的菌株进行了成功建立和验证。在一个包含 758 株近期结核分枝杆菌分离株的集合中确定的 spoligotype 分布与 SITWITWEB 国际数据库中海地分离株的先前数据一致，这些数据是使用传统的基于膜的方法获得的。在目前的形式下， spoligotyping 可能适合作为资源有限国家结核分枝杆菌基因分型的高通量一线工具。
