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Abstract
Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (>94%). The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.
Highlights
The detection of clinically relevant viral pathogens is an essential task performed by medical microbiology laboratories, to help establish diagnosis, guide the subsequent treatment and contribute to public health surveillance, including monitoring of emerging agents
polymerase chain reaction (PCR) is used in the screening and detection of numerous infectious viral or bacterial species, by amplifying the target nucleic acids extracted from patient sample, over several orders of magnitude [1,2]
This approach allows for one step detection and quantification of extracted nucleic acid and it is an excellent example of PCR technology that illustrates the feasible integration of microfluidics into such commercial systems
Summary
The detection of clinically relevant viral pathogens is an essential task performed by medical microbiology laboratories, to help establish diagnosis, guide the subsequent treatment and contribute to public health surveillance, including monitoring of emerging agents. The droplet digital PCR (ddPCRTM, Bio-Rad, Hercules, CA, USA) system commercially available from Bio-Rad incorporates the close-channel microfluidic based droplet generation method to create a large library (~15,000) of sub-microliter droplets, which are dispensed from a large PCR sample/reagent mixture (~20 μL) [16] This approach allows for one step detection and quantification of extracted nucleic acid and it is an excellent example of PCR technology that illustrates the feasible integration of microfluidics into such commercial systems. We have modified the previously designed continuous, D-DEP electrode architecture for the PCR thermal cycling to produce a spatially multiplexed PCR micro-device, suitable for carrying out several different qRT-PCR reactions in parallel (up to eight assays per chip) and with a built-in flexibility to accommodate different cycling parameters for each reaction The performance of this micro-device is illustrated by the parallel execution of assays for the detection of Influenza A virus and Influenza B virus in different panels of clinical samples. The reported multiplexed qRT-PCR assays are a first demonstration of a D-DEP based DMF device for analysing multiple clinically relevant viral pathogens in panels of extracted patient samples
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