Spinning Disk Platform for Microfluidic Digital Polymerase Chain Reaction
University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112, USA.Analytical Chemistry (Impact Factor: 5.64). 02/2010; 82(4):1546-50. DOI: 10.1021/ac902398c
An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.
- "Die kommerziellen Plattformen werden durch die entsprechenden Firmen vermarktet. Alle anderen sind Forschungsplattformen der Forschergruppen Rödiger , Shen , Sundberg  und Morrison . "
Article: Digitale PCR in der Labordiagnostik[Show abstract] [Hide abstract]
ABSTRACT: [Article in German] The need for new highly sensitive, cost-efficient, fast and robust nucleic acid detection and quantification technologies is a driving force. PCR, especially quantitative PCR (qPCR), is the method of choice in diagnostics and life-sciences. The digital PCR (dPCR) provides a new technology to measure absolute quantities of nucleic acids without the need for calibration curves. This review gives details on the dPCR technology and available platforms. We discuss the platform differences, common features as well as their advantages and disadvantages. http://www.biospektrum.de/sixcms/media.php/1093/12268-015-0610-y.pdfBioSpektrum 09/2015; 05(21):507-510. DOI:10.1007/s12268-015-0610-y
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- "Therefore, the utility of U-dHRM in diagnostic and research applications will be improved by future work to increase the number and reduce the volumes of digital reactions, leading to improved resolution in the presence of contaminants, higher content, higher throughput and reduced reagent costs. High-throughput microfluidic digital droplet technologies (8,43–46) that incorporate simultaneous highly controlled heating and sensitive fluorescence detection for millions of reactions are needed. In a real heterogeneous sample where unknown sequences are expected and starting concentrations of targets may be unknown, millions of broad-based U-dHRM reactions will ensure enough dynamic range for successful single molecule detections (Table 3). "
ABSTRACT: Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.Nucleic Acids Research 08/2013; 41(18). DOI:10.1093/nar/gkt684 · 9.11 Impact Factor
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- "We used capillary PCR and a PCR melting approach for preliminary characterization of extraction output and establishment of RT-PCR protocols. The RT-PCR disks were loaded first by pipetting 10 µL each of PCR mix and mineral oil (M5904, Sigma-Aldrich Corporation), dyed with Oil Red O (Matheson Coleman & Bell, Gardena, CA), into the individual loading reservoirs of the disk and the disk was then spun at 4000 rpm for 5 min to move the fluid from the middle to the outside of the disk . Disk images before and after the PCR runs were taken and samples were centrifuged into collecting vials and subjected to melting or gel electrophoresis as required. "
ABSTRACT: This work reports design and characterization results for an integrated microfluidics-based biosensor being developed for field-based Foot-and-Mouth-Disease Virus (FMDV) detection. This paper outlines the specific challenges towards integration of such a device and describes coating materials and characterization of the different modules using appropriate viral analogues. The modules include: a sample preparation module for viral RNA extraction using a disposable silica filter and an on-chip RT PCR module for detection with high sensitivity and specificity. INTRODUCTION FMDV is highly contagious and prevalent in the most economically important animals worldwide . FMDV exists in seven different serotypes, the early identification of which is important in understanding the disease and its potential spread in the population. Our laboratory is developing a field-based analysis tool for rapid FMDV serotype identification from clinical samples that range from vesicular fluid to feet or nose epithelium to whole blood. The overall goal of this project is to create an integrated system with sample in-answer out capability. The requirement of distinguishing different serotypes poses a significant challenge towards automation and total integration due to the inherent complexity and potential contamination issues. This necessitates an amplification approach that is capable of rapidly processing multiple samples at once without any cross-talk. For this purpose our initial work has used an automated microfluidic extraction unit (Figure 1) with a spinning disk platform (Figure 2) for RT-PCR that will allow us to analyze the different FMDV serotypes with high sensitivity in less than three hours with our current analysis protocol. The current version of the RT-PCR disk contains 5 separate channels with individual loading chambers for different samples. Figure 1 is a photograph of the microfluidic extraction system in its current format. The top portion consists of a PDMS microfluidic platform complete with on-chip valves (31 pneumatic valves), reservoir pumps (9 chambers that also work as pumps), and a disposable extraction filter. The system is controlled by a LabView program that operates a sequence of solenoid valves to run the RNA extraction protocol, which uses the same chemical sequence as the Qiagen RNeasy Mini spin kit. FABRICATION Extraction chamber module: The microfluidic chip for flow control with on-chip microvalves was fabricated using three-layers: a fluid channel layer, a membrane, and a valve control channel layer. A thin membrane layer serves as the valve and pump actuator separating the fluid channel layer and a pneumatic valve control channel layer . The fluid and control microchannel layers are molded in PDMS using xurographically-patterned tapes as the mold. An off-the-shelf thin silicone sheet was used as the membrane layer [Bisco HT 6135, Rogers Corp, CT]. The layers were bonded together by activating the surfaces with a corona discharge treatment. As the final step, access holes for valves, inlets and outlets were cored into the molded PDMS to allow fluidic connections to the fluid and control channels. The on-chip pneumatic valves are actuated by applying vacuum to the control channel layer. Spinning disk RT-PCR module: The disk for RT-PCR uses centrifugal force to move fluid into 5 short microfluidic channels with individual inlets located at the center to run separate chemistries for different viral serotypes. The disk for RT-PCR is fabricated from thin film polycarbonates to create an inexpensive disposable, requiring only centrifugation for fluid control. The entire disk is 120 mm in diameter, the size of a CD, and is 375 m thick (see Figure 2). The channel layer disks were manufactured using the process of xurography [3, 4].
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