Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Anuj, S. N., Whiley, D. M., Kidd, T. J., Ramsay, K. A., Bell, S. C., Syrmis, M. W., Grimwood, K., Wainwright, C. E., Nissen, M. D. and Sloots, T. P. (2011) Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis. Clinical Microbiology and Infection, 17 9: 1403-1408. doi:10.1111/j.1469-0691.2010.03439.x


Author Anuj, S. N.
Whiley, D. M.
Kidd, T. J.
Ramsay, K. A.
Bell, S. C.
Syrmis, M. W.
Grimwood, K.
Wainwright, C. E.
Nissen, M. D.
Sloots, T. P.
Title Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis
Formatted title
Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis
Journal name Clinical Microbiology and Infection   Check publisher's open access policy
ISSN 1198-743X
1469-0691
Publication date 2011-09
Sub-type Article (original research)
DOI 10.1111/j.1469-0691.2010.03439.x
Volume 17
Issue 9
Start page 1403
End page 1408
Total pages 6
Place of publication Oxford, United Kingdom
Publisher Wiley-Blackwell Publishing
Collection year 2012
Language eng
Abstract Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.
Keyword Clonal
Cystic fibrosis
Pseudomonas aeruginosa
Single-nucleotide polymorphism
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
School of Medicine Publications
Clinical Medical Virology Centre Publications
 
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