Journal
JOURNAL OF MOLECULAR DIAGNOSTICS
Volume 9, Issue 5, Pages 639-644Publisher
AMER SOC INVESTIGATIVE PATHOLOGY, INC
DOI: 10.2353/jmoldx.2007.070012
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Funding
- NIGMS NIH HHS [GM 077780, R43 GM077780] Funding Source: Medline
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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.
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