Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection.
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.
Goldmeyer
James
J
BioHelix Corporation, 32 Tozer Road, Beverly, MA 01915, USA.
Kong
Huimin
H
Tang
Wen
W
D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics*; D048348:Reverse Transcription
D004268:DNA-Binding Proteins; D012367:RNA, Viral; D004265:DNA Helicases