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


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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  1. 1.

    Chin CD, Linder V, Sia SK. Lab-on-a-chip devices for global health: past studies and future opportunities. Lab Chip. 2007;7:41–57.

  2. 2.

    Drancourt M, Michel-lepage A, Boyer S. The point-of-care laboratory in clinical microbiology. Clin Microbiol Rev. 2016;29:429–47.

  3. 3.

    St John A, Price CP. Existing and emerging technologies for point-of-care testing. Clin Biochem Rev. 2014;35:155–67.

  4. 4.

    Pai NP, Vadnais C, Denkinger C, Engel N, Pai M. Point-of-care testing for infectious diseases: diversity, complexity, and barriers in low- and middle-income countries. PLoS Med. 2012;9:e1001306.

  5. 5.

    Bissonnette L, Bergeron MG. Diagnosing infections—current and anticipated technologies for point-of-care diagnostics and home-based testing. Clin Microbiol Infect. 2010;16:1044–53.

  6. 6.

    Peeling RW, Mabey D. Point-of-care tests for diagnosing infections in the developing world. Clin Microbiol Infect. 2010;16:1062–9.

  7. 7.

    Kapasi AJ, Dittrich S, Gonzalez IJ, Rodwell TC. Host biomarkers for distinguishing bacterial from non-bacterial causes of acute febrile illness: a comprehensive review. PLoS One. 2016;11:1–29.

  8. 8.

    Crump J, Gove S, Parry C. Management of adolescents and adults with febrile illness in resource limited areas. BMJ. 2011;343:18.

  9. 9.

    Crump JA, Morrissey AB, Nicholson WL, Massung RF, Stoddard RA, Galloway RL, et al. Etiology of severe non-malaria febrile illness in northern Tanzania: a prospective cohort study. PLoS Negl Trop Dis. 2013; 7.

  10. 10.

    World Health Organization. Antimicrobial resistance. http://www.who.int/mediacentre/factsheets/fs194/en/ (2016). Accessed 1 Jan 2016.

  11. 11.

    Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. PT. 2015;40:277–83.

  12. 12.

    Dittrich S, Tadesse BT, Moussy F, Chua A, Zorzet A, Tängdén T, et al. Target product profile for a diagnostic assay to differentiate between bacterial and non-bacterial infections and reduce antimicrobial overuse in resource-limited settings: an expert consensus. PLoS One. 2016;11:e0161721.

  13. 13.

    Mohd Hanafiah K, Garcia M, Anderson D. Point-of-care testing and the control of infectious diseases. Biomark Med. 2013;7:333–47.

  14. 14.

    Nouvellet P, Garske T, Mills HL, Nedjati-Gilani G, Hinsley W, Blake IM, et al. The role of rapid diagnostics in managing Ebola epidemics. Nature. 2015;528:S109–16.

  15. 15.

    World Health Organization. Ebola virus disease. 2016. http://www.who.int/mediacentre/factsheets/fs103/en/ (2016). Accessed 22 May 2016.

  16. 16.

    Centers for Disease Control and Prevention. Ebola virus disease. http://www.cdc.gov/vhf/ebola/diagnosis/ (2015). Accessed 1 Sep 2016.

  17. 17.

    World Health Organization. Laboratory diagnosis of Ebola virus disease: interim guideline. Geneva: World Health Organization; 2014.

  18. 18.

    Fernández-Carballo BL, McGuiness I, McBeth C, Kalashnikov M, Borrós S, Sharon A, et al. Low-cost, real-time, continuous flow PCR system for pathogen detection. Biomed Microdevices. 2016;18:34.

  19. 19.

    Park S, Zhang Y, Lin S, Wang T-H, Yang S. Advances in microfluidic PCR for point-of-care infectious disease diagnostics. Biotechnol Adv. 2012;29:830–9.

  20. 20.

    Zhang C, Xu J, Ma W, Zheng W. PCR microfluidic devices for DNA amplification. Biotechnol Adv. 2006;24:243–84.

  21. 21.

    Wallow TI, Morales AM, Simmons BA, Hunter MC, Krafcik KL, Domeier LA, et al. Low-distortion, high-strength bonding of thermoplastic microfluidic devices employing case-II diffusion-mediated permeant activation. Lab Chip. 2007;7:1825–31.

  22. 22.

    Silva RM, Pratas D, Castro L, Pinho AJ, Ferreira PJSG. Three minimal sequences found in Ebola virus genomes and absent from human DNA. Bioinformatics. 2015;31:2421–5.

  23. 23.

    Jun SR, Leuze MR, Nookaew I, Uberbacher EC, Land M, Zhang Q, et al. Ebolavirus comparative genomics. FEMS Microbiol Rev. 2015;39:764–78.

  24. 24.

    Ren K, Zhou J, Wu H. Materials for microfluidic chip fabrication. Acc Chem Res. 2013;46:2396–406.

  25. 25.

    Epigem. UV embossing. http://epigem.co.uk/technology/uv-embossing (2016). Accessed 11 Jan 2016.

  26. 26.

    SVG Optronics. R2R UV nanoimprinting. http://en.svgoptronics.com/cp/html/?32.html (2016). Accessed 11 Jan 2016.

  27. 27.

    Foundation for Innovative New Diagnostics. Situational review of Ebola diagnostics and opportunities for rapid improvement. Geneva: Foundation for Innovative New Diagnostics; 2014.

  28. 28.

    Sauer-Budge AF, Mirer P, Chatterjee A, Klapperich CM, Chargin D, Sharon A. Low cost and manufacturable complete microTAS for detecting bacteria. Lab Chip. 2009;9:2803–10.

  29. 29.

    Chatterjee A, Mirer PL, Zaldivar Santamaria E, Klapperich C, Sharon A, Sauer-Budge AF. RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation. Anal Chem. 2010;82:4344–56.

  30. 30.

    Prakash R, Pabbaraju K, Wong S, Wong A, Tellier R, Kaler K. Multiplex, quantitative, reverse transcription PCR detection of influenza viruses using droplet microfluidic technology. Micromachines. 2014;6:63–79.

  31. 31.

    Kaler K, Prakash R. Droplet microfluidics for chip-based diagnostics. Sensors. 2014;14:23283–306.

  32. 32.

    Beer NR, Wheeler EK, Lee-Houghton L, Watkins N, Nasarabadi S, Hebert N, et al. On-Chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets. Anal Chem. 2008;80:1854–8.

  33. 33.

    Li Y, Zhang C, Xing D. Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection. Microfluid Nanofluid. 2011;10:367–80.

  34. 34.

    Obeid PJ, Christopoulos TK. Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection. Anal Chim Acta. 2003;494:1–9.

  35. 35.

    Yamanaka K, Saito M, Kondoh K, Hossain MM, Koketsu R, Sasaki T, et al. Rapid detection for primary screening of influenza a virus: microfluidic RT-PCR chip and electrochemical DNA sensor. Analyst. 2011;136:2064–8.

  36. 36.

    Hartung R, Brösing A, Sczcepankiewicz G, Liebert U, Häfner N, Dürst M, et al. Application of an asymmetric helical tube reactor for fast identification of gene transcripts of pathogenic viruses by micro flow-through PCR. Biomed Microdevices. 2009;11:685–92.

  37. 37.

    Felbel J, Reichert A, Kielpinski M, Urban M, Häfner N, Dürst M, et al. Technical concept of a flow-through microreactor for in-situ RT-PCR. Eng Life Sci. 2008;8:68–72.

  38. 38.

    Asiello PJ, Baeumner AJ. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip. 2011;11:1420–30.

  39. 39.

    Ahmad F, Hashsham SA. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review. Anal Chim Acta. 2012;733:1–15.

  40. 40.

    Niemz A, Ferguson TM, Boyle DS. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 2011;29:240–50.

  41. 41.

    Tang Y-W, Ou C-Y. Past, present and future molecular diagnosis and characterization of human immunodeficiency virus infections. Emerg Microbes Infect. 2012;1:e19.

  42. 42.

    Yan L, Zhou J, Zheng Y, Gamson AS, Roembke BT, Nakayama S, et al. Isothermal amplified detection of DNA and RNA. Mol BioSyst. 2014;10:970–1003.

  43. 43.

    Towner JS, Sealy TK, Ksiazek TG, Nichol ST. High-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings. J Infect Dis. 2007;196:S205–12.

  44. 44.

    Trombley AR, Wachter L, Garrison J, Buckley-Beason VA, Jahrling J, Hensley LE, et al. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and new world hantaviruses. Am J Trop Med Hyg. 2010;82:954–60.

  45. 45.

    Weidmann M, Mühlberger E, Hufert FT. Rapid detection protocol for filoviruses. J Clin Virol. 2004;30:94–9.

  46. 46.

    Towner JS, Rollin PE, Bausch DG, Sanchez A, Crary SM, Vincent M, et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome. J Virol. 2004;78:4330–41.

  47. 47.

    Kreuels B, Wichmann D, Emmerich P, Schmidt-Chanasit J, de Heer G, Kluge S, et al. A case of severe Ebola virus infection complicated by gram-negative septicemia. N Engl J Med. 2014;371:2394–401.

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