Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

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.