Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies

Abstract

A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology. Amplified fragment length polymorphism analysis, originally developed for typing crop plants, consists of a simple restriction-ligation reaction and a subsequent PCR amplification. In a single-step reaction, the genomic DNA is digested and the restriction fragments are ligated to specially constructed adapters. PCR amplification of such tagged restriction fragments with primers complementary to the adapters allows the detection of restriction fragment length polymorphisms upon resolution on agarose gels. The method is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. The accuracy of the method was tested by comparison with standard restriction fragment length polymorphism typing performed with both a ribosomal and a genomic probe.

Nosocomial Legionella pneumophila serogroup 5 outbreak associated with persistent colonization of a hospital water system

“…AFLP analysis was carried out according to the original method described by Valsangiacomo et al (21) using AFLPPstI-G as a selective primer.…”

Combination of pulsed-field gel electrophoresis (PFGE) and single-enzyme amplified fragment length polymorphism (SAFLP) for differentiation of multiresistant Salmonella enterica serotype typhimurium

“…Selective amplification by PCR of sets of these fragments is achieved using primers corresponding to the contiguous base sequences in the adapter restriction site plus one or more nucleotides in the original target DNA. The adapters are designed with an additional base pair in the restriction site [13], so that after ligation to the restricted fragment, the base pair can be eliminated. The resulting PCR-amplified DNA fragments may then be analyzed by polyacrylamide gel electrophoresis (PAGE) or by agarose gel electrophoresis (AGE).…”

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

“…AFLP analysis involves digestion of cellular DNA with two restriction enzymes; ligation of speci¢c oligonucleotide adaptors to the pertinent restriction sites; PCR ampli¢cation using primers speci¢c to the relevant restriction site/adaptor sequence; and separation and detection of the PCR-ampli¢ed fragments. Several studies have shown that AFLP is a highly sensitive method for characterising a wide range of bacterial pathogens, including Legionella pneumophila [11], Aeromonas spp. [10], and Salmonella spp.…”

“…[9]. However, AFLP requires optimisation for di¡erent organisms to account for genomic di¡erences (notably genome size and G+C content), and also to ensure the resolving power of the electrophoretic detection system employed is used to its full potential [10,11]. Here we describe a method suitable for AFLP ¢ngerprinting of C. jejuni and C. coli and evaluate its potential for molecular epidemiological studies of these taxa.…”