1887

Abstract

Laboratory detection of spp., in particular aeruginosa, remains an important assay in the management of patients with cystic fibrosis (CF). As the and genes of have now been cloned and their nucleotide sequences determined, the aim of this study was to develop a novel PCR assay for the detection of spp. from patients with CF by employing conserved primer regions of the heat-shock protein domain gene. A PCR assay was designed that targeted a 536 bp region of the gene to detect spp. PCR amplification of genomic DNA from extracted organisms generated an amplicon of the expected size (approx. 536 bp) for all ( = 60), putida, fluorescens and stutzeri isolates examined, but did not produce a positive amplicon for several other genera and species that are commonly isolated from the sputum of CF patients. RFLP analysis of the amplicons of all isolates demonstrated a single RFLP type that consisted of three bands at approximately 80, 190 and 250 bp; direct sequencing of the amplicons demonstrated the presence of a single sequence type, indicating the highly conserved nature of this region. In addition, the assay successfully produced a positive signal from primary non-selective plates of three known culture-positive CF patients, but was unable to generate a signal in a further six CF patients who had no history of infection with or other spp. This assay is recommended to detect the presence of spp., including , from primary culture plates that originate from laboratory analysis of CF patients’ sputum, particularly at review, in those patients with no previous history of infection or those who appear to be transiently colonized by this organism. Employment of such molecular methodologies, in conjunction with routine clinical sputum cultures, may provide improved information on the microbial status of CF patients, which will aid clinicians in both optimum patient management in terms of antibiotic regimes and CF centre infection-control practices.

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2003-09-01
2019-11-19
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