Poster Y14 - Multiplexed point-of-care device for the simultaneous detection and of IAV, IBV, SARS-CoV-2 and RSV from nasal swab samples

Introduction: The recent COVID-19 pandemic demonstrated the limits of today’s diagnostic infrastructure, but promoted innovation in both academia and industry to develop more rapid, efficient, and sensitive detection modalities. Yet, there has been less attention given to respiratory viruses such as Influenza A/B, and Respiratory Syncytial Virus (RSV) that share many symptoms/transmission routes and have co-infection incidence with SARS-CoV-2. These four viruses are commonly detected using the multiplexed ‘gold standard’ reverse transcriptase-polymerase chain reaction (RT-PCR), whose limitations include the need for trained laboratory personnel, delayed sample-to-answer time, expensive instrumentation, reliance on cold-chain supply for reagents, and lengthy extraction and purification of the viruses.

Thus, there is a need for a point-of-care accessible, rapid, sensitive, and user-friendly-device that simultaneously detects these respiratory viruses. To achieve this goal, this team developed a microfluidic device combining chemical viral nucleic acid extraction, Reverse Transcriptase-Loop Mediated Isothermal Amplification (RT-LAMP), and smart phone image capture. Fluorescence image analysis via our app gives virus identification and possibility of quantification within 30 minutes, enabling rapid detection of the four viral targets as well as a negative and positive control deployable in resource-limited healthcare systems, or at home.

Materials and

Methods: We first spiked a nasal swab with synthetic nasal fluid contaminated with IAV, IBV, RSV, and/or SARS-CoV-2 viral particles. Following that, the nasal swab was immersed in 0.5% Tw20, resulting in a 50x dilution of the virus. Combining this virus-positive lysis buffer to liquid LAMP reagent mix in a 1:7 ratio, then taking 25 µL of this mixture to PCR tubes containing dried target-specific LAMP primers and placing these tubes in a benchtop thermocyler for 60°C 60 min, represented our off-cartridge processing protocol to determine our assay’s sensitivity. The on-cartridge protocol only differed in the final step where we pipetted the sample+liquid reagent mix to the snap in tube of our cartridge (Fig. 1a).

The 3D-printed microfluidic cartridge has the following elements: 1.) The sample inlet tube. 2.) The suction pump. 3.) The reconstitution region that can contain six dried reagent pellets if desired over liquid reagents. 4.) The serpentine mixing region. 5.) 6-well detection region: 6 wells contain pre-dried-spotted LAMP primers for each virus target and the MS2 bacteriophage positive control, 1 well with no dried primers for the negative control.

This cartridge is placed into a reader, which contains a heater supplying a 60°C constant temperature for the LAMP reaction, lenses, and LEDs for image capture. On the reader, a slot for a smartphone app to capture (1/min) and analyze images for positive or negative fluorescence signals.

Results, Conclusions, and Discussions: Results and

Discussion: We have successfully developed 4 target virus off- and on-cartridge LAMP assays using a spiked nasal swab as the sample. This protocol uses nasal fluid and nasal swab, to ensure the robustness and reliability of our method considering complexity of biological samples and its application as a true point-of-care device. Our nasal swab protocol had the following off-cartridge sensitivities: 5x104 TCID50/mL active IAV, 105 CEID50/mL active IBV, 5x103 copies/µL heat inactivated SARS-CoV-2, and 105 TCID50/mL active RSV (Fig. 1b). On-cartridge, we tested swabs spiked with low and high concentrations of virus, as well as positive and negative controls. Representative smartphone-collected fluorescent images of the reaction reservoirs shown in Fig. 1c.

Conclusion: This work describes the development and optimization of a multiplexed point-of-care system for the rapid and easy detection and identification of four respiratory viruses: IAV, IBV, SARS-CoV-2, and RSV from a nasal swab. Overall, this multiplexed technology can improve the rapidness and efficiency of the respiratory virus clinical testing workflow. The ease of use of the device, combined with the quick chemical extraction, dried primers, and facile results interpretation, provides a point-of-care solution for the simultaneous detection of these viruses. Future directions in this field would include simultaneous detection of both disease serological markers and virion nucleic acids on one microfluidic chip.

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