Novel high-speed real-time PCR method (Hyper-PCR): Results from its application to adenovirus diagnosis

Abstract

PCR, including real-time PCR, usually requires at least 1 h to obtain results. To shorten this time, a novel real-time PCR method (Hyper-PCR) was developed. This method utilizes high-speed DNA polymerase and a thin disc-type reaction vessel that can quickly alter the temperature of the reaction mixture in a newly developed PCR machine. Reactions capable of amplifying adenovirus (Ad) DNA can be completed within 11 min (3 temperature steps, 55 cycles). Hyper-PCR can detect 3.1-18.0 DNA copies/reaction of Ad types 1-4, 7, 8, 11, 15, 19, and 37. Hyper-PCR and conventional real-time PCR were applied to diagnose 147 clinical samples, and the results were compared. Hyper-PCR had a sensitivity of 100% (73/73) and a specificity of 100% (74/74), using conventional real-time PCR as the gold standard. Our newly developed PCR method, Hyper-PCR, was able to diagnose Ad infection within 17 min (not including the time for genome extraction). The thermocycling time of the novel PCR is faster than that of previously available PCR applications, and this method is thought to be potentially applicable to clinical and environmental diagnostics, where rapid diagnosis is important.