Rapid and accurate detection of Pseudomonas aeruginosa by real-time polymerase chain reaction with melting curve analysis targeting gyrB gene

Abstract

Laboratory detection of Pseudomonas spp., particularly Pseudomonas aeruginosa, is an important assay in the nosocomial control. The study was designed firstly to establish a new assay-applied LightCycler polymerase chain reaction (PCR) technology with melting curve analysis (MCA). A total of 224 Gram-negative isolates were used to verify the assay system. The PCR with MCA method using the P. aeruginosa-specific gyrase B gene primers was rapid and accurate; the total run is approximately 3 h, and the sensitivity and specificity relative to the Vitek (bioMerieux, Hazelwood, MO) results were 98.1% and 100%, respectively. Vitek identification system was not able to identify the isolates from the new Pseudomonas otitidis spp. opposite to the real-time PCR. This assay was validated to be accurate with an overall sensitivity and specificity of 98.7% and 98.9%, respectively. Conclusively, this rapid and accurate PCR assay with MCA will help to manage and control infections with P. aeruginosa.

POM-1 metallo-β-lactamase–producing Pseudomonas otitidis isolate from a patient with chronic otitis media

“…molecular characterization among 104 phenotypically identified Pseudomonas aeruginosa isolates from otic infections in Japan (Motoshima et al, 2007). We report a case of chronic otitis media due to P. otitidis.…”

Melting Curve Analysis for Rapid Detection of Topoisomerase Gene Mutations inHaemophilus influenzae

“…FQ-resistant H. influenzae often carries mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and the parC genes, which encode subunits of DNA gyrase and topoisomerase IV, respectively (9,11,16,18,23). A combination of real-time PCR methods and melting curve analysis (PCR-MCA) is a useful tool for the rapid detection of key gene mutations associated with drug resistance in various microorganisms (13,24,25), but there are no reports about H. influenzae. The aim of this study was to develop a PCR-MCA method for detecting H. influenzae strains by targeting a total of four QRDR positions in the gyrA (codons 84 and 88) and the parC (codons 84 and 88) genes that are frequently associated with FQ resistance (9,11,16,23).…”

Detection and Quantification of Pathogenic Bacteria and Fungi Using Real-Time Polymerase Chain Reaction by Cycling Probe in Patients With Corneal Ulcer

“…While the pathogen in 1 of the 3 cases with discrepant findings (cases 21-23) ( Table 5) was thought to be Acanthamoeba (case 21) (Table 5), the other 2 cases (cases [22][23] were cured by multidrug therapy and the causative pathogen could not be verified. Eleven cases had positive PCR results only (2 fungal and 9 bacterial pathogens) ( Table 5).…”

“…The selected target genes for the specific pathogens were Cap5G (GeneBank U81973) and Cap8G (GeneBank U73374) for S aureus, 21 LytA (Takara Bio Inc) for S pneumoniae, gyrB (GeneBank EF064840.1) for P aeruginosa, 22 mecA (GeneBank X52593) for methicillin-resistant S aureus, 23,24 ITS2 (GeneBank ABO32174) for Candida species, 25 and EF1-alpha (GeneBank DQ247583) for Fusarium species. 26 All the probes were labeled with a 6-carboxyfluorecein reporter dye at the 5Ј end and with an Eclipse Quencher dye (Epoch Biosciences, Bothell, Washington) at the 3Ј end.…”