Antonishyn N A, McDonald R R, Chan E L, Horsman G, Woodmansee C E, Falk P S, Mayhall C G
Saskatchewan Health, Provincial Laboratory, Regina, Saskatchewan S4S 5W6, Canada.
J Clin Microbiol. 2000 Nov;38(11):4058-65. doi: 10.1128/JCM.38.11.4058-4065.2000.
Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the "gold standard" for molecular typing in epidemiology.
基于荧光的扩增片段长度多态性分析（fbAFLP）是一种基于对限制性片段扩增子集进行荧光分析的新型检测方法。fbAFLP检测涉及从基因组DNA的完全酶切产物中对限制性片段进行选择性PCR扩增。将带有引物特异性位点的接头与含有选择性核苷酸的引物连接起来，在保持限制性内切酶分析优势的同时，充分发挥了PCR的潜力。通过聚丙烯酰胺凝胶电泳进行基于荧光的片段分析，可提供同源性评估所需的准确条带大小测定。fbAFLP获得的大量系统发育信息特征非常适合聚类分析和数据库开发。该方法具有出色的可重复性，操作和解读都很简便。我们通过fbAFLP对一所大学医院爆发疫情中30株流行病学特征明确的耐万古霉素肠球菌分离株进行了分型。fbAFLP数据的聚类与流行病学、微生物学及脉冲场凝胶电泳数据相匹配。本研究证明了fbAFLP在流行病学调查中具有前所未有的实用性。标准化和自动化方面的未来发展将使fbAFLP成为流行病学分子分型的“金标准”。
