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Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection

Abstract

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide.

Photograph of the RT-PCR thermoplastic chip

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Acknowledgements

The work presented is based on activities related to the international research project ML2 - Multilayer MicroLab (grant agreement no. 318088). The ML2 project was funded by the European Commission within the Seventh Framework Programme. The authors thank Affymetrix Inc. for providing the custom-made master mix. Dolomite Microfluidics is gratefully acknowledged for providing microfluidic connectors.

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Correspondence to Alexis F. Sauer-Budge.

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The authors declare that they have no competing interests.

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Fernández-Carballo, B.L., McBeth, C., McGuiness, I. et al. Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection. Anal Bioanal Chem 410, 33–43 (2018). https://doi.org/10.1007/s00216-017-0689-8

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Keywords

  • Lab on a chip
  • Quantitative reverse transcription polymerase chain reaction
  • RNA-based virus detection
  • Infectious diseases
  • Point of care
  • Ebola virus