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Journal of Clinical Microbiology, September 2003, p. 4312-4317, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4312-4317.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis

Xuan Qin,1 Julia Emerson,2 Jenny Stapp,3 Lynn Stapp,3 Patrick Abe,3 and Jane L. Burns3,4*

Department of Laboratory Medicine, Divisions of,1 Pulmonology,2 Infectious Disease, Department of Pediatrics,4 Therapeutics Development Network Resource Center for Microbiology, Children's Hospital and Regional Medical Center, University of Washington, Seattle, Washington3

Received 3 March 2003/ Returned for modification 23 April 2003/ Accepted 19 June 2003

Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.

* Corresponding author. Mailing address: 4800 Sand Point Way N.E. 8G-1, Seattle, WA 98105. Phone: (206) 987-2073. Fax: (206) 987-3890. E-mail: jane.burns{at}seattlechildrens.org.

Journal of Clinical Microbiology, September 2003, p. 4312-4317, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4312-4317.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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