Browsing by Subject "pcr"

Background The diagnosis of sepsis is challenging because the infection can be caused by more than 50 species of pathogens that might exist in the bloodstream in very low concentrations, e.g., less than 1 colony-forming unit/ml. As a result, among the current sepsis diagnostic methods there is an unsatisfactory trade-off between the assay time and the specificity of the derived diagnostic information. Although the present qPCR-based test is more specific than biomarker detection and faster than culturing, its 6 ~ 10 hr turnaround remains suboptimal relative to the 7.6%/hr rapid deterioration of the survival rate, and the 3 hr hands-on time is labor-intensive. To address these issues, this work aims to utilize the advances in microfluidic technologies to expedite and automate the ``nucleic acid purification - qPCR sequence detection'' workflow.

Methods and Results This task is evaluated to be best approached by combining immiscible phase filtration (IPF) and digital microfluidic droplet actuation (DM) on a fluidic device. In IPF, as nucleic acid-bound magnetic beads are transported from an aqueous phase to an immiscible phase, the carryover of aqueous contaminants is minimized by the high interfacial tension. Thus, unlike a conventional bead-based assay, the necessary degree of purification can be attained in a few wash steps. After IPF reduces the sample volume from a milliliter-sized lysate to a microliter-sized eluent, DM can be used to automatically prepare the PCR mixture. This begins with compartmenting the eluent in accordance with the desired number of multiplex qPCR reactions, and then transporting droplets of the PCR reagents to mix with the eluent droplets. Under the outlined approach, the IPF - DM integration should lead to a notably reduced turnaround and a hands-free ``lysate-to-answer'' operation.

As the first step towards such a diagnostic device, the primary objective of this thesis is to verify the feasibility of the IPF - DM integration. This is achieved in four phases. First, the suitable assays, fluidic device, and auxiliary systems are developed. Second, the extent of purification obtained per IPF wash, and hence the number of washes needed for uninhibited qPCR, are estimated via off-chip UV absorbance measurement and on-chip qPCR. Third, the performance of on-chip qPCR, particularly the copy number - threshold cycle correlation, is characterized. Lastly, the above developments accumulate to an experiment that includes the following on-chip steps: DNA purification by IPF, PCR mixture preparation via DM, and target quantification using qPCR - thereby demonstrating the core procedures in the proposed approach.

Conclusions It is proposed to expedite and automate qPCR-based multiplex sparse pathogen detection by combining IPF and DM on a fluidic device. As a start, this work demonstrated the feasibility of the IPF - DM integration. However, a more thermally robust device structure will be needed for later quantitative investigations, e.g., improving the bead - buffer mixing. Importantly, evidences indicate that future iterations of the IPF - DM fluidic device could reduce the sample-to-answer time by 75% to 1.5 hr and decrease the hands-on time by 90% to approximately 20 min.