Evaluation of different primers for DNA fingerprinting of Legionella pneumophila serogroup 1 strains by polymerase chain reaction.
A DNA fingerprinting method for the characterization of Legionella pneumophila serogroup 1 strains was established. This method was based on the DNA extraction using Chelex 100 and subsequent PCR analysis using primers under conditions of low stringency. Sixteen single primers were tested for the typing of the 10 epidemiologically unrelated reference strains of L. pneumophila serogroup 1 as well as patient isolates and environmental strains isolated from the water system of a hospital where patients with legionellosis were treated. In addition, a combination of two primers (Lpm-1 and Lpm-2) originally established for the specific detection of Legionella strains was tested. The PCR results were compared with two further subtyping methods, i.e. monoclonal antibody analysis and pulsed-field gel electrophoresis. The type strains Philadelphia 1, Knoxville 1, Allentown 1, Benidorm 0303E, Bellingham 1, and France 5811 could be distinguished clearly in experiments using all of the primers. Depending on the primer used, Heysham 1 and Oxford 4032E showed different DNA profiles. The strains Olda and Camperdown 1 were nearly indistinguishable. In contrast, the analysis by PFGE and MAb subtyping revealed distinct types for all 10 reference strains. The discrimination of the patient isolates from two suspected cases of nosocomial legionellosis and environmental isolates was not possible with the 16 single primers used in the study. However, the PCR assay with the combination of Lpm-1 and Lpm-2 as well as the PFGE and MAb analysis were able to differentiate distinct types. The use of the sequence-specific primers under low-stringency annealing conditions allowed both simultaneous gene detection as well as epidemiological typing of Legionella strains.
Wiese
Jutta
J
Medical Institute of General Hygiene and Environmental Health, Göttingen, Germany. jwiese@ifm-geomar.de
Helbig
Jürgen H
JH
Lück
Christian
C
Meyer
Heinz-Georg W
HG
Jansen
Bernd
B
Dunkelberg
Hartmut
H
D015373:Bacterial Typing Techniques; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004783:Environmental Microbiology; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification* / Q000235:genetics; D007877:Legionnaires' Disease / Q000453:epidemiology* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012703:Serotyping
D017931:DNA Primers; D004269:DNA, Bacterial