Development And Application Of Integration And Multiplex Methods Of Digital PCR Microfluidic Chips

Digital PCR technology based on microfluidic technology has developed rapidly in the past decade.Due to its unique advantages and industrialization prospects,it has been widely used in early cancer diagnosis,pathogen detection,and prenatal diagnosis.Although digital PCR has shown outstanding advantages in precise quantification,its integration and multiplexing development has been restricted.The integration of the digital PCR chip can realize the accurate quantitative method of "sample in-digital-result out",which improves detection efficiency,ccuracy and stability of the detection results.Multiplex digital PCR is beneficial to improve the efficiency,economy and system of the detection process.This dissertation focuses on the integration and multiplexing of digital PCR chips and has carried out preliminary applications.The main research contents are as follows:(1)For the integrated development of digital PCR,a rapid nucleic acid extraction method was established and a polytetrafluoroethylene tube based nucleic acid extraction system that can be integrated with digital PCR chips was developed.The system uses polytetrafluoroethylene tube loaded with reagents to extract nucleic acids by using the two-phase interfacial tension of water and oil in combination with the highly efficient magnetic bead method for nucleic acid extraction.The nucleic acid is extracted by using PCR mix as the elution system,and the obtained nucleic acid can be directly transferred to the digital PCR chip for reaction after elution.The mineral oil phase is used for interfacial separation,and it can be used for physical separation directly into the digital PCR chip after the nucleic acid extraction.The system can complete nucleic acid extraction from cells and blood within 5 minutes and integrate directly with the digital PCR chip,so as to accurately quantify nucleic acid molecules and achieve the "sample-in-digital-answer-out" detection method,which is of great application value in rapid nucleic acid extraction and the integration of digital PCR chips.(2)In order to realize the integration and multiplex detection simultaneously,a microfluidic chip with integrated nucleic acid extraction and multiplexed digital RPA was developed and applied to the detection of various pathogenic bacteria.The magnetic bead method for nucleic acid extraction and the special chip design allow for efficient separation of nucleic acid extraction reagents from a single oil phase.Four primer probes were pre-embedded on the digital RPA chip using a screw valve and a separately designed sample inlet.The chip is capable of performing nucleic acid extraction in 15 minutes and amplification detection in 30 minutes,and the entire detection process takes only 45 minutes.We successfully detected three pathogens from a simulated sample(milk)using this chip,which enables the detection of "sample in-digital-answer-out".(3)In order to further improve the multiplexed detection performance of digital PCR chips,achieve higher-order digital PCR detection and reduce the processing difficulty of the chips,a new multiplex digital PCR chip is developed in this paper.The chip utilizes a separately designed sample inlet,based on the self-absorption dispensing principle,to achieve the digital PCR detection of six targets using monochromatic fluorescence without any external pumping structure.The chip is made of polydimethylsiloxane and has six detection zones and a four-layer structure.The chip demonstrated accurate quantitative performance by using known concentrations of standards and real-time digital PCR.The accuracy of the chip’s multiplexe detection performance was demonstrated by comparing the results of five EGFR mutation site standards with those of a commercial digital PCR assay.Finally,as a proof-of-concept,we successfully detected EGFR mutation sites in 15 lung cancer patients,demonstrating the potential clinical value of the chip.In summary,this dissertation focuses on the integration and multiplexing of digital microfluidic chips,develops a nucleic acid extraction method for integration of digital PCR chips,realizes the integrated and multiplex detection of digital detection chips,and develops a high-level multiplex digital PCR chip and carries out preliminary applications.