A rapid PFGE protocol for typing Legionella isolates from fresh or frozen samples.
Summary of "A rapid PFGE protocol for typing Legionella isolates from fresh or frozen samples."
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.
Affiliation
Bioinformatics Unit, Center for Advanced Studies, Research and Development in Sardinia (CRS4), Polaris, Ed. 1, 09010, Pula, Italy, orsini@crs4.it.
Journal Details
This article was published in the following journal.
Name: Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy
ISSN: 1437-7780
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/22526389
- DOI: http://dx.doi.org/10.1007/s10156-012-0416-5
Medical and Biotech [MESH] Definitions
Bacterial Typing Techniques
Procedures for identifying types and strains of bacteria. The most frequently employed typing systems are BACTERIOPHAGE TYPING and SEROTYPING as well as bacteriocin typing and biotyping.
Legionella Longbeachae
A species of gram-negative, aerobic bacteria in the genus LEGIONELLA, first isolated in Long Beach, California. Infection is associated with the use of potting SOIL.
Bacteriophage Typing
A technique of bacterial typing which differentiates between bacteria or strains of bacteria by their susceptibility to one or more bacteriophages.
Multilocus Sequence Typing
Direct nucleotide sequencing of gene fragments from multiple housekeeping genes for the purpose of phylogenetic analysis, organism identification, and typing of species, strain, serovar, or other distinguishable phylogenetic level.
Sperm Motility
Movement characteristics of SPERMATOZOA in a fresh specimen. It is measured as the percentage of sperms that are moving, and as the percentage of sperms with productive flagellar motion such as rapid, linear, and forward progression.
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