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One‐step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase
Author(s) -
Kranaster Ramon,
Drum Matthias,
Engel Nicole,
Weidmann Manfred,
Hufert Frank T.,
Marx Andreas
Publication year - 2010
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200900200
Subject(s) - thermus aquaticus , taq polymerase , reverse transcriptase , taqman , biology , microbiology and biotechnology , hot start pcr , nuclease , multiple displacement amplification , polymerase , primer dimer , polymerase chain reaction , dna , polymerase chain reaction optimization , real time polymerase chain reaction , nested polymerase chain reaction , genetics , multiplex polymerase chain reaction , dna extraction , gene
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.