title: A rapid PFGE protocol for typing Legionella isolates from fresh or frozen samples.
abstract: Within the realm of studies on nosocomial infections and epidemiology, pulsed field gel electrophoresis (PFGE) is often considered as the "gold standard" for typing of Legionella or other bacteria. Performing this protocol usually requires 2-5 days; this excessive time requirement, lack of a standardized procedure, and high cost have limited its use. However, recently the typing of Legionella with PFGE has been reduced to about 2 days, and we further shortened the procedure by reducing the time for the plug preparation and electrophoresis steps. To shorten plug preparation, we used a strong thermal shock and high-temperature washes to reduce cell lysis and DNA isolation time, and we stressed the electrophoresis to obtain comparable macrorestriction patterns among strains in 16 h. The DNA digestion phase was not altered. We also applied this protocol directly to frozen bacteria from strain collections with the aim of shortening the entire procedure. We developed a protocol that can be completed in 24 h, or less if necessary, while avoiding some typical artifacts of traditional procedures. This new protocol also provides good results when directly applied to frozen material from strain collections, thus saving bacteria growth time. Our observations indicate that PFGE tolerates a wide range of adjustments, allowing its application to fresh or frozen samples in a short amount of time.
authors.affiliation: Bioinformatics Unit, Center for Advanced Studies, Research and Development in Sardinia (CRS4), Polaris, Ed. 1, 09010, Pula, Italy. orsini@crs4.it
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Orsini
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Romano-Spica
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D006801:Humans; D007875:Legionella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D007876:Legionellosis / Q000382:microbiology*; D013048:Specimen Handling