Centrifugal microfluidic system for primary amplification and secondary real-time PCR.

Pre-amplification is a basis for numerous polymerase chain reaction (PCR) protocols but bears severe contamination risks due to handling of high-copy DNA samples. Therefore we developed a self-contained centrifugal microfluidic system comprising pre-stored reagents; it enables pre-amplification of specific DNA sequences prior to automated aliquoting and real-time PCR in a modified commercial thermocycler.

Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform.

We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (microTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 microm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 microl (CV 3.4%, N = 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.

Chair/bedside diagnosis of oral and respiratory tract infections, and identification of antibiotic resistances for personalised monitoring and treatment.

Global healthcare systems are struggling with the enormous burden associated with infectious diseases, as well as the incessant rise of antimicrobial resistance. In order to adequately address these issues, there is an urgent need for rapid and accurate infectious disease diagnostics. The H2020 project DIAGORAS aims at diagnosing oral and respiratory tract infections using a fully integrated, automated and user-friendly platform for physicians' offices, schools, elderly care units, community settings, etc. Oral diseases (periodontitis, dental caries) will be detected via multiplexed, quantitative analysis of salivary markers (bacterial DNA and host response proteins) for early prevention and personalised monitoring. Respiratory Tract Infections will be diagnosed by means of DNA/RNA differentiation so as to identify their bacterial or viral nature. Together with antibiotic resistance screening on the same platform, a more efficient treatment management is expected at the point-of-care. At the heart of DIAGORAS lies a centrifugal microfluidic platform (LabDisk and associated processing device) integrating all components and assays for a fully automated analysis. The project involves an interface with a clinical algorithm for the comprehensive presentation of results to end-users, thereby increasing the platform's clinical utility. DIAGORAS' performance will be validated at clinical settings and compared with gold standards.