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9 June 2017 A miniaturized total analysis system for real-time PCR (Conference Presentation)
Hidenori Nagai
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Proceedings Volume 10215, Advanced Environmental, Chemical, and Biological Sensing Technologies XIV; 102150V (2017) https://doi.org/10.1117/12.2267096
Event: SPIE Commercial + Scientific Sensing and Imaging, 2017, Anaheim, CA, United States
Abstract
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.
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Hidenori Nagai "A miniaturized total analysis system for real-time PCR (Conference Presentation)", Proc. SPIE 10215, Advanced Environmental, Chemical, and Biological Sensing Technologies XIV, 102150V (9 June 2017); https://doi.org/10.1117/12.2267096
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KEYWORDS
Microfluidics
Luminescence
Polymers
Switching
Biosensing
Current controlled current source
Environmental sensing
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