The gold standard for molecular detection and identification of bacteria and fungi is PCR and DNA sequencing of ribosomal RNA genes. PCR and DNA sequencing (PCRS) has been the mainstay for the identification of selected bacterial and fungal cultures in our laboratory since 2003. In addition, PCRS has been effectively used for the detection and identification of microbial pathogens direct in patient samples leading to a rapid diagnosis of a bacterial or fungal aetiology. Our current system using conventional PCR targets 16S and ITS regions for bacteria and fungi respectively. This approach utilises agarose gel electrophoresis for PCR product detection and to estimate the PCR product concentration prior to sequencing. However, agarose gel electrophoresis is time consuming, operators are exposed to ethidium bromide, requires significant post-PCR handling of PCR products and results can be subjective.
In this investigation we report a universal real time (RT) PCR assay using the Roche LightCycler 480 as an alternative to conventional PCR and gel electrophoresis. In addition, we report the advantages RT PCR compared to conventional PCR and the use of Crossing Points (Cp) to determine the PCR product concentration as an alternative to gel electrophoresis. We compared staining intensity (K-Value) with Cp and sequencing results for the controls, 100 clinical isolates of fungi and 50 bacterial isolates. We determined that RT PCR Cp values were a reliable measure to estimate PCR product concentration. Identification of all isolates was concordant between conventional PCR and RT PCR. The real time approach is more rapid, less labour intensive and better suited to routine diagnostic use for the identification of bacterial and fungal cultures. Furthermore this approach is well-suited for the rapid detection of bacterial and fungal DNA in clinical samples.