Affiliation:
1. Medical Technology Research Centre, Faculty of Health, Medicine and Social Care Anglia, Ruskin University, Chelmsford CB1 1PT, UK
2. Shipley Consulting, Vancouver, WA 98682, USA
Abstract
Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called “FlashPCR”, uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications.
Funder
National Institute for Health Research
Reference40 articles.
1. Simultaneous amplification and detection of specific DNA sequences;Higuchi;Bio/Technology,1992
2. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions;Higuchi;Bio/Technology,1993
3. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis;Bustin;Clin. Sci.,2005
4. Improving the analysis of quantitative PCR data in veterinary research;Bustin;Vet. J.,2012
5. Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses?;Lopez;Curr. Issues Mol. Biol.,2009