FlashPCR: Revolutionising qPCR by Accelerating Amplification through Low ∆T Protocols

• Published in: International journal of molecular sciences 2024-02, Vol.25 (5), p.2773
• Main Authors: Bustin, Stephen A, Kirvell, Sara, Nolan, Tania, Shipley, Gregory L
• Format: Article
• Language: English
• Subjects: COVID-19; Denaturation; Efficiency; molecular diagnostics; Pathogens; point of care; Proteins; Protocol; qPCR; reverse transcription; Severe acute respiratory syndrome coronavirus 2; Temperature
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms25052773

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.