title: Diagnostics method for the rapid quantitative detection and identification of low-level contamination of high-purity water with pathogenic bacteria.
abstract: High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 10(3) cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) and 3.7 GE, respectively. This analysis demonstrated that it was possible to detect a spiked bacterial load of 1.06 × 10(2) cfu/100 ml for P. aeruginosa and 2.66 × 10(2) cfu/100 ml for B. cepacia from a 200-ml filtered HPW sample. The rapid diagnostics method described can reliably detect, identify, and quantify low-level contamination of HPW with P. aeruginosa and the Burkholderia genus in <4 h. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of HPW, medical devices, and patient-care equipment.
authors.affiliation: Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Minogue
authors.forename: Kate
authors.initials: K
authors.lastname: Reddington
authors.forename: Siobhan
authors.initials: S
authors.lastname: Dorai-Raj
authors.forename: Nina
authors.initials: N
authors.lastname: Tuite
authors.forename: Eoin
authors.initials: E
authors.lastname: Clancy
authors.forename: Thomas
authors.initials: T
authors.lastname: Barry
mesh_terms: D019117:Burkholderia / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D013010:Sonication; D013997:Time Factors; D014871:Water Microbiology; D018508:Water Purification; D060753:Water Quality / Q000592:standards
chemical_list: D004269:DNA, Bacterial