Rapid typing of influenza viruses using super high-speed quantitative real-time PCR

Sakurai, Akira; Nomura, Namiko; Nanba, Reiko; Sinkai, Takayuki; Iwaki, Tsunehito; Obayashi, Taminori; Hashimoto, Kazuhiro; Hasegawa, Michiya; Sakoda, Yoshihiro; Naito, Akihiro; Morizane, Yoshihito; Hosaka, Mitsugu; Tsuboi, Kunio; Kida, Hiroshi; Kai, Akemi; Shibasaki, Futoshi

The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(−1) plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.