Raspberry Pi-Based Real-Time Digital PCR System Development And Application Verification

Digital polymerase chain reaction(dPCR)has become one of the mainstream technology for detecting and quantifying nucleic acids.Fluorescence detection is a commonly used method for digital PCR endpoint detection.Since the method cannot achieve correct classification of evaporation,non-specific amplification,and low reaction efficiency chambers,the accuracy of quantitative results of digital PCR is affected.The emergence of real-time digital PCR platform provides an effective way to improve the quantitative accuracy of digital PCR.Aiming at the characteristics of the self-priming digital PCR chip developed by our group,a small-size,low-cost real-time digital PCR system has been successfully developed in this thesis.The main work in this thesis is listed as follows:1.A Raspberry Pi-based real-time PCR system was designed and built.The microfluidic dPCR chip,the thermocycler,the fluorescence image setup and the Raspberry Pi with the touch screen were integrated in this system.To our knowledge,it is the smallest and lowest cost real-time digital PCR platform.2.A chamber array-based chip's real-time fluorescence detection and analysis system was developed,whose microchambers classification results are more specific and accurate compared the end-point detection.3.The PCR experiment of template DNA was carried out to verify the accuracy and stability of the system.And the comparative experiment carried out later on further proved that the real-time digital PCR was exhibited with more ideal value,with a better digital PCR quantitative accuracy.4.Three kinds of chamber array-based chip's end-point fluorescence image was collected and one type of droplet-based chip's fluorescence image processing was finished in our system,indicating that the system we developed has strong compatibility and scalability.This system realized the development of high integrated and low-cost real-time digital PCR system.It will effectively improve the accuracy of digital PCR quantification and shows a potential to be an alternative for real-time dPCR assays.