A mutated thermostable Thermus aquaticus DNA polymerase with reverse transcriptase activity for one step RNA pathogen detection
Résumé
We describe the cloning and characterisation of a mutated thermostable DNA polymerase from Thermus aquaticus (Taq) which 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. Indeed, we found comparable detection sensitivities compared to commercial available RT-PCR systems without further optimization of reaction parameters thus making this enzyme highly suitable for any PCR probe based RNA detection method.
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