Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens.
We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.
Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Samuel
S
Yang
Shin
S
Lin
Gabor D
GD
Kelen
Thomas C
TC
Quinn
James D
JD
Dick
Charlotte A
CA
Gaydos
Richard E
RE
Rothman
D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D001424:Bacterial Infections / Q000382:microbiology*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D005374:Filtration / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D019914:Taq Polymerase
D015342:DNA Probes; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase