Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis.
Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing: (i) the fingerprints of 16 colonies of a single H. influenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. influenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. influenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. influenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H. influenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.
van Belkum
A
A
Diagnostisch Centrum SSDZ, Department of Molecular Biology, Delft, The Netherlands.
Duim
B
B
Regelink
A
A
Möller
L
L
Quint
W
W
van Alphen
L
L
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D001483:Base Sequence; D016384:Consensus Sequence; D003550:Cystic Fibrosis / Q000382:microbiology; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D005838:Genotype; D006192:Haemophilus Infections / Q000382:microbiology; D006193:Haemophilus influenzae / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D006801:Humans; D008173:Lung Diseases, Obstructive / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D013183:Sputum / Q000382:microbiology
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial