Direct DNA and RNA detection from blood for the detection of bacterial pathogens Abstract: Real time polymerase chain reaction (PCR) and real time reverse transcription polymerase chain reaction (RT PCR) are now increasingly used in clinical microbiology laboratories for the detection of bacterial pathogens from clinical specimens. A major problem with real time PCR and RT PCR based diagnostic assays of clinical specimens is the low sensitivity and even false negative results caused by PCR inhibitors which are ubiquitous in clinical specimens. Consequently a variety of sample processing methods PCR facilitators and specific PCR buffers have been developed for successful DNA and RNA detection from clinical specimens. However sample processing methods are generally time consuming labor intensive not suitable for automation and also have the potential of losing target molecules. Meanwhile PCR facilitators and specific PCR buffers are generally sample specific involve the risk of contamination need further standardization and may not be equally effective under different laboratory conditions.<br>An alternative to using sample processing methods PCR facilitators and specific PCR buffers is the employment of a sturdy DNA polymerase which is resistant to PCR inhibitors for direct DNA and RNA detection from clinical specimens. Thermus thermophilus (Tth) polymerase has proven to be resistant to several common PCR inhibitors present in clinical specimens for DNA detection and also exhibits reverse transcriptase activity in the presence of Mn ions. However the capacity of Tth polymerase which acts as both reverse transcriptase and DNA polymerase for RNA detection in the presence of various clinically relevant PCR inhibitors has not been investigated in detail. In this thesis 14 PCR inhibitors originating from blood urine feces bodily fluids muscle tissues and reagents used during nucleic acid extraction are employed to evaluate the capacity of Tth polymerase for RNA detection. The results show that these PCR inhibitors have different inhibitory effects on the real time RT PCR assays by Tth polymerase and the inhibitory effects are concentration dependent. Furthermore the capacity of Tth polymerase for RNA detection in the presence of various PCR inhibitors is better or at least comparable to its reported capacity for DNA detection in the presence of the same PCR inhibitors. As a result RNA may be directly analyzed in the presence of some co purified PCR inhibitors or even directly from certain crude clinical specimens su ...