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.
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 such as urine and blood by Tth polymerase.
After testing the capacity of Tth polymerase for RNA detection in the presence of various clinically relevant PCR inhibitors, this enzyme is used to directly detect exogenous bacterial DNA and RNA from large volumes of whole human blood. Anticoagulants of EDTA, citrate, and heparin in blood specimens can cause PCR inhibition by depleting DNA polymerase co-factors (i.e., Mg ions and Mn ions), especially in the assays with high concentrations of blood. Therefore, the concentrations of Mg ions and Mn ions are increased to compensate for this inhibitory effect. In combination with optimized concentrations of Mg ions (6 mM) and Mn ions (4 mM), Tth polymerase enables efficient detection of exogenous bac terial DNA and RNA from 50% (v/v) and 40% (v/v) blood, respectively. Blood
specimens treated with various anticoagulants, collected from different healthy individuals, stored under different conditions are also investigated, which show no
significant influence on the capacity of Tth polymerase for DNA and RNA detection. The detection limit of DNA from 10-30% (v/v) blood is 5.8 copies/uL and that for RNA from 10-40% (v/v) blood is 6800 copies/uL. The detection limit of bacteria targeting DNA or RNA is both 6.6 CFU/uL.
Compared with the reported methods or commercially available kits, the Tth polymerase-based method combined with optimized concentration of Mg ions is capable of detecting DNA from a higher concentration of whole blood (up to 50%), which allows direct detection of a lower concentration of bacterial pathogens from a blood specimen. Furthermore, direct RNA detection from up to 40% (v/v) has been achieved using Tth polymerase combined with optimized concentration of Mn ions. To the best of our knowledge, direct RNA detection from whole blood has not been reported. This enables direct detection of bacterial pathogens targeting certain RNA molecules which naturally exist in a high copy number in a bacterium, allowing the detection of a lower concentration of bacterial pathogens

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2019

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2020
• Creator: Cai, Dongyang
• Contributor: Urban, Gerald A. ; Albert-Ludwigs-Universität Freiburg. Fakultät für Angewandte Wissenschaften

Further information

• DOI: 10.6094/UNIFR/154619
• URN: urn:nbn:de:bsz:25-freidok-1546199
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 1:52 PM CET