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Digital PCR development
Gaňová, Martina ; Lipov,, Jan (oponent) ; Jansová,, Eva (oponent) ; Neužil, Pavel (vedoucí práce)
In recent years, microtechnological and nanotechnological methods have proven to be a powerful analytical tools for deoxyribonucleic acid (DNA) analysis. There are reviewed different techniques developed over the last decade to amplify the nucleic acids (NA), including microfluidic systems. Polymerase chain reaction (PCR) is widely used in molecular biology to amplify target NA in vitro. The number of groups working on PCR worldwide is significant given the substantial social and economic impact of this technique, for example, in medical diagnostic, criminology, food processing, or environmental studies. Nowadays, the coronavirus disease 2019 pandemic proved the importance of developing more accessible technologies for diagnosing viral diseases. The thesis presents the development of two versions of PCR platforms for NA detection, droplet real-time quantitative PCR (qPCR) and digital PCR (dPCR). The critical components of both platforms were fabricated using the microtechnological procedures for surfaces modification and lithographic fabrication, allowing the development of hydrophobic cover glasses or silicon microchips. The results of the thesis demonstrate the design, assembly, and testing with optimization of both platforms. The PCR technology consists of a software part controlled by the LabView program and a hardware part consisting of a temperature control system and a fluorescence imaging system. The droplet qPCR was conducted in 0.3 µL of the master mix droplet containing the target gene encapsulated with 2 µL of mineral oil. The droplets were pipetted on the hydrophobic cover glass, placed on the thermoelectric cooler (TEC) under the fluorescence microscope to conduct thermal cycling. The fluorescence changes during thermal cycling were captured by photomultiplier tubes and monitored by oscilloscope. The results of the testing also present the multiplexing capability of the developed technique. Three synthetic genes using intercalating fluorescent dye for simultaneous detection and quantification based on a single fluorescence channel were introduced. The droplet qPCR technology was a crucial platform for further development of the dPCR platform. The dPCR platform employed a silicon microchip with microwell sample dispersion to the 26 448 microwells, each with a target diameter of 50 µm and a volume of 59 pL. The microchip loaded with the master mix containing the target DNA was covered by the mineral oil and cover glass modified by polydimethylsiloxane and Parylene C. The heating/cooling system of thermal cycling with the TEC was similar to the droplet qPCR platform. The fluorescent imaging system used a complementary metal-oxide-semiconductor (known as CMOS) camera to capture the fluorescent images. The developed dPCR was demonstrated for applications in human medical research. The synthetic virus DNA, isolated virus DNA, and female genomic DNA were tested. The thesis reveals the development of the dPCR system, which is a part of a new dPCR technique that is more affordable, easy to use with simple sample delivery, which are the most common problems why it has not yet found much popularity among laboratories. The silicon-based microchip dPCR improved the system performance due to a large number of wells. The employment of such dPCR benefits of high sensitivity, low signal to noise ratio, accuracy or lower detection limit, and multiplexing capability.
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