title: Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.
abstract: Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.
authors.affiliation: Multi-drug Resistant Organism Repository and Surveillance Network (MRSN), Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Clifford
authors.forename: Michael
authors.initials: M
authors.lastname: Milillo
authors.forename: Jackson
authors.initials: J
authors.lastname: Prestwood
authors.forename: Reyes
authors.initials: R
authors.lastname: Quintero
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Zurawski
authors.forename: Yoon I
authors.initials: YI
authors.lastname: Kwak
authors.forename: Paige E
authors.initials: PE
authors.lastname: Waterman
authors.forename: Emil P
authors.initials: EP
authors.lastname: Lesho
authors.forename: Patrick
authors.initials: P
authors.lastname: Mc Gann
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000378:metabolism; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S