A miniaturized total analysis system for real-time PCR (Conference Presentation)

The microfluidic device for the handheld RT-qPCR thermal cycler was attached with three heaters. The temperatures of these heaters were constantly controlled to 42˚C, 96˚C and 56˚C for reverse transcription, denaturization, and anealing/extension, respectively. The PCR solution was injected with about 20 µl and flowed in the microchannel repeatedly by a switching of two pumps. The two kinds of fluorescence for FAM and ROX were measured on a detection point placed between the two heaters for the denaturization and the anealing/extension. For the microfluidic device, we have developed the handheld RT-qPCR thermal cycler. The size of the system is 200 x 100 x 50 mm and the weight is only 0.6 kg including dry-cell batteries. As the target for the high-speed microfluidic RT-qPCR, influenza A virus was examined. Reverse transcription and qPCR were carried out sequentially in the same microchannel as a one-step RT-qPCR. As the results of 45 cycles with 3 s for denaturation and 9 s annealing/extension after 30 s for RT and 10s for activation of DNA polymerase, influenza A virus could be detected within only 12 minutes, and the detection limit was approximately 50 pfu/ml.