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SCIENCE CHINA Chemistry, Volume 63 , Issue 10 : 1498-1506(2020) https://doi.org/10.1007/s11426-020-9800-6

A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing

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  • ReceivedMay 7, 2020
  • AcceptedJun 23, 2020
  • PublishedJul 27, 2020

Abstract

The outbreak of virus-induced infectious diseases poses a global public-health challenge. Nucleic acid amplification testing (NAAT) enables early detection of pandemic viruses and plays a vital role in preventing onward transmission. However, the requirement of skilled operators, expensive instrumentation, and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients. Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive, specific, and rapid viral nucleic acid testing. The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification (RT-LAMP) were integrated into the reaction units of a microfluidic disc. The whole processing steps such as injection of reagents, fluid actuation by rotation, heating and temperature control, and detection of fluorescence signals were carried out automatically by a customized instrument. We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) armored RNA particles. The estimated limit of detection for armored RNA particles is 2 copies per reaction, the throughput is 21 reactions per disc, and the assay sample-to-answer time is approximately 70 min. This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol, and can be readily adapted for virus detection outside the diagnostic laboratory.


Funded by

the National Natural Science Foundation of China(91959101,21904028)

Chinese Academy of Sciences(YJKYYQ20180055,YJKYYQ20190068,ZDBS-LY-SLH025)

and the Strategic Priority Research Program of Chinese Academy of Sciences(XDB36000000)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (91959101, 21904028), Chinese Academy of Sciences (YJKYYQ20180055, YJKYYQ20190068, ZDBS-LY-SLH025), and the Strategic Priority Research Program of Chinese Academy of Sciences (XDB36000000).


Interest statement

The authors declare no conflict of interest.


Supplement

The supporting information is available online at http://chem.scichina.com and http://link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Overview of the automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing. (a) Procedures of viral nucleic acid testing by integrating the release of nucleic acids and reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection within a microfluidic disc. Scale bar, 1 cm. (b) Schematic of the customized instrument for automated sample-to-answer detection (color online).

  • Figure 2

    RT-LAMP for detecting N, E and O genes of SARS-CoV-2. (a) The primer set for N gene. Photographs of RT-LAMP in tubes using 106/μL SARS-CoV-2 armored RNA particles with N gene and N gene primer set (b), E gene and E gene primer set (c) and O gene and E gene primer set (d). All samples generated signals appearing as green after amplification. Tris buffer was used as NC. The primers and PC-RNA in the commercial kit were used as PC. Scale bar, 1 cm (color online).

  • Figure 3

    Colorimetric and fluorescent detection of N gene in armored RNA particles at different concentrations of 1–104 copies/μL using the N primer set. Tris buffer was used as NC. Scale bar, 1 cm (color online).

  • Figure 4

    Centrifugal microfluidic disc for viral nucleic acid testing. (a) Layer-by-layer structure of the microfluidic disc. From bottom to top: Channel layer I, Channel layer II, and the sealing layer. Scale bar, 2 cm. (b) The structure of reaction unit consisting of 3 chambers. From disc center to edge: Chamber I for reagent injection, Chamber II for sample injection, and Chamber III for nucleic acid release and RT-LAMP reaction. Scale bar, 0.5 cm. (c) Step-by-step illustration of viral nucleic acid release, amplification, and detection within the integrated microfluidic disc (color online).

  • Figure 5

    CFD simulation of an impact-induced mixing between sample and reagent solutions at 3000 r/min. Scale bar, 3 mm (color online).

  • Figure 6

    Characterization of customized, stand-alone instrument. (a) Components of the instrument; (b) accuracy test for reagent injection; (c) rotation stability test; (d) temperature control test; (e) signal collection test (color online).

  • Figure 7

    Highly sensitive detection of viral nucleic acids using the automated instrument. (a) The amplification curve of SARS-CoV-2 armored RNA particles with N gene (0.5, 1, 10, 102, 103 copies/μL) inside the microfluidic disc. Tris buffer was used as NC. (b) The amplification curve of plasmids containing N gene of SARS-CoV or SARS-CoV-2 (102 copies/μL). Water was used as NC (color online).

  • Figure 8

    Automated microfluidic system for viral detection using oropharyngeal swab samples. (a) Procedures of sample collection and viral nucleic acid testing. (b) The amplification curve of oropharyngeal swab samples from healthy volunteer and spiked with SARS-CoV-2 armored RNA particles (0.5, 103 copies/μL). Tris buffer was used as NC (color online).

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