In the clinical setting, molecular microbiologic detection methods have classically focused on a single microorganism or a small group of microorganisms. Broad range molecular detection of bacteria in clinical specimens is an approach that parallels a conventional culture-based approach, may be more rapid, and offers the possibility of identifying so-called "non-culturable" organisms. A common molecular target used for broad range detection of bacteria using polymerase chain reaction (PCR) is 16S ribosomal DNA (rDNA). Primers are designed based on areas of 16S rDNA sequence conserved amongst bacteria; sequencing of the amplified DNA yields data which can be used to identify the source organism. Our research collaborator, Dr. Robin Patel, at Mayo Clinic, Rochester, MN, has extensive experience with this approach. Unfortunately, this technique is not applicable to the direct diagnosis of polymicrobial bacterial infections unless the mixed 16S rDNA amplification products can be separated and then separately sequenced. Traditional approaches to separation of mixed amplification products include temperature gradient gel electrophoresis, denaturing gradient gel electrophoresis, and cloning; these approaches are cumbersome, especially for use in the routine diagnostic setting. The purpose of this proposal is to evaluate a new application for separation of mixed 16S rDNA amplification products prior to sequencing - denaturing high performance liquid chromatography using the WAVE(r) System (Transgenomic Inc., Omaha NE). This system has been shown to be able to separate PCR products differing by a single nucleotide. The sensitivity of this rapid, high-throughput technique to detect bacteria present in varying ratios in mixed species populations will be assessed. Denaturing high performance liquid chromatography-based profiling and sequence-based investigation of 16S rDNA (or alternate broad range target) PCR amplification products could be applicable to a wide variety of patient specimen types (e.g. synovial fluid, cerebrospinal fluid, blood, abscess material) and diseases (e.g. prosthetic joint infection, meningitis, bacteremia), as well as to characterization of mixed-species bacterial biofilms, normal flora (e.g. gastrointestinal tract, genital tract, oral cavity) and agricultural or environmental specimens.
Thesaurus Terms: bacterial DNA, method development, nucleic acid quantitation /detection, ribosomal DNA high throughput technology, nucleic acid sequence high performance liquid chromatography, polymerase chain reaction
