Desai M, Tanna A, Wall R, Efstratiou A, George R, Stanley J
Molecular Biology Unit, Central Public Health Laboratory, London NW9 5HT, United Kingdom.
J Clin Microbiol. 1998 Nov;36(11):3133-7. doi: 10.1128/JCM.36.11.3133-3137.1998.
Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was carried out for an outbreak of group A streptococcal (GAS) invasive disease. Streptococcal genomic DNAs were digested with endonucleases EcoRI and MseI, site-specific adaptors were ligated, and PCR amplification was carried out with an EcoRI adaptor-specific primer labelled with fluorescent dye. Amplified fragments of up to 600 bp in size were separated on a polyacrylamide sequencing gel which contained internal size markers in each lane. These data were automatically scanned and analyzed, fragments were precisely sized (+/-1 bp), and electropherograms were generated for each genome with GeneScan 2.1 software. All isolates were compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP profiles, compared with 12 macrorestriction profiles by pulsed-field gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of invasive disease in a care-of-the-elderly system. It provided an automated analysis of the whole genome of bacterial isolates. It was reproducible, more discriminatory, and capable of higher throughput than other molecular typing methods. Given agreed conditions, FAFLP would be reproducible between laboratories for rapid characterization of outbreak strains.
对一起 A 组链球菌（GAS）侵袭性疾病暴发进行了荧光扩增片段长度多态性（FAFLP）分析。用核酸内切酶 EcoRI 和 MseI 消化链球菌基因组 DNA，连接位点特异性接头，并用荧光染料标记的 EcoRI 接头特异性引物进行 PCR 扩增。大小达 600 bp 的扩增片段在每条泳道都含有内部大小标记的聚丙烯酰胺测序凝胶上进行分离。这些数据被自动扫描和分析，片段精确测定大小（±1 bp），并使用 GeneScan 2.1 软件为每个基因组生成电泳图谱。所有分离株都以这种方式进行比较。在所检测的 27 株 GAS 分离株中，我们发现了 18 种 FAFLP 图谱，而脉冲场凝胶电泳显示有 12 种宏观限制性图谱。FAFLP 很容易区分导致老年护理系统中侵袭性疾病暴发的 GAS 血清型 M77 的两个克隆的基因型。它提供了对细菌分离株全基因组的自动化分析。与其他分子分型方法相比，它具有可重复性、更高的鉴别力且通量更高。在给定的一致条件下，FAFLP 在不同实验室之间具有可重复性，可用于快速鉴定暴发菌株。
