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One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase

One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase

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KRANASTER, Ramon, Matthias DRUM, Nicole ENGEL, Manfred WEIDMANN, Frank T. HUFERT, Andreas MARX, 2010. One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase. In: Biotechnology Journal. 5(2), pp. 224-231. ISSN 1860-6768. eISSN 1860-7314. Available under: doi: 10.1002/biot.200900200

@article{Kranaster2010-02Onest-12607, title={One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase}, year={2010}, doi={10.1002/biot.200900200}, number={2}, volume={5}, issn={1860-6768}, journal={Biotechnology Journal}, pages={224--231}, author={Kranaster, Ramon and Drum, Matthias and Engel, Nicole and Weidmann, Manfred and Hufert, Frank T. and Marx, Andreas} }

Marx, Andreas We describe the cloning and characterization of a mutated thermostable DNA polymerase from Thermus aquaticus (Taq) that exhibits an increased reverse transcriptase activity and is therefore designated for one-step PCR pathogen detection using established real-time detection methods. We demonstrate that this Taq polymerase mutant (Taq M1) has similar PCR sensitivity and nuclease activity as the respective Taq wild-type DNA polymerase. In addition, and in marked contrast to the wild-type, Taq M1 exhibits a significantly increased reverse transcriptase activity especially at high temperatures (>60°C). RNA generally hosts highly stable secondary structure motifs, such as hairpins and G-quadruplexes, which complicate, or in the worst case obviate, reverse transcription (RT). Thus, RT at high temperatures is desired to weaken or melt secondary structure motifs. To demonstrate the ability of Taq M1 for RNA detection of pathogens, we performed TaqMan probe-based diagnostics of Dobrava viruses by one-step RT-PCR. We found similar detection sensitivities compared to commercially available RT-PCR systems without further optimization of reaction parameters, thus making this enzyme highly suitable for any PCR probe-based RNA detection method. First publ. in: Biotechnology Journal 5 (2010), 2, pp. 224-231 2010-02 Engel, Nicole Engel, Nicole eng One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase Weidmann, Manfred deposit-license Drum, Matthias Marx, Andreas Hufert, Frank T. 2011-06-16T09:14:53Z Kranaster, Ramon Hufert, Frank T. 2011-06-16T09:14:53Z Weidmann, Manfred Drum, Matthias Kranaster, Ramon

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