Quantitative Multiprobe PCR Assay for Simultaneous Detection and Identification to Species Level of Bacterial Pathogens

Abstract

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.Currently, the standard method for diagnosing the presence of bacterial pathogens in clinical samples relies on culture techniques. However, active research is under way using new molecular methods to decrease detection time and increase assay sensitivity. PCR has emerged as the molecular method of choice in achieving these objectives. The utility of PCR and other molecular methods is evidenced by the recent guidelines issued by the NCCLS in 1999 encouraging the use of such methods in clinical laboratories performing bacterial identification assays (11).To detect the presence of any bacterial pathogen in a clinical sample, primers annealing to regions of DNA conserved across a wide range of bacterial genomes have been employed. The design of such universal primers has often focused on the 16S rRNA gene (17). The presence of multiple copies of this gene within the bacterial genome facilitates its amplification by PCR. Further, sufficient sequence variability allows phylogenetic information to be attained for the purposes of microbial identification. However, up to the present, assays which provide for both universal detection and species identification require a second post-PCR processing step, which can be technically cumbersome and slow the time to reporting of results (9, 14).Universal PCR-based bacterial detection systems have also been hampered by contamination issues. High sequence conservation of the DNA region chosen for PCR primer annealing coupled with the immense amplification power of PCR results in the amplification of exceedingly minor bacterial contaminants, leading to false positives. Attempts to decontaminate PCR materials have involved nearly all known methods to destroy DNA, including UV irradiation, 8-MOP treatment, and incubation with various enzymes, such as DNase, restriction enzymes, or both in combination (2, 4). Thus far, none of these methods has been shown to be entirely effective or reproducible.Assessment of bacterial contamination can most reliably be made using real-time detection methods to characterize PCR amplification. Briefly, real-time PCR am...

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