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J Mol Diagn. 2007 Nov;9(5):639-44.

Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection.

Author information

1
BioHelix Corporation, 32 Tozer Road, Beverly, MA 01915, USA.

Abstract

The high complexity and cost of polymerase chain reaction-based molecular diagnostics sometimes limits their use in the clinical diagnostics setting. A new helicase-based isothermal amplification method offers an alternative to standard polymerase chain reaction, allowing amplification and detection of specific DNA sequences at a constant reaction temperature without thermocycling equipment. Herein, we describe the development of a novel one-tube isothermal reverse transcription-thermophilic helicase-dependent amplification (RT-tHDA) platform for RNA target detection based on the already established tHDA system. The RT-tHDA platform is highly sensitive and specific for a variety of RNA targets tested, including purified RNA molecules, armored RNA particles, and RNA virus. Moreover, rapid one-step RT-tHDA can be achieved by inclusion of an extreme thermostable single-stranded DNA binding protein in the reaction, resulting in one millionfold amplification of Ebola virus-armored RNA in less than 10 minutes. This RT-tHDA method expands on the known methods to amplify specific RNA targets and results in an easily prepared and contained platform.

PMID:
17975029
PMCID:
PMC2049050
DOI:
10.2353/jmoldx.2007.070012
[Indexed for MEDLINE]
Free PMC Article

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