Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses

Abstract

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is still spreading around the globe causing immense public health and socioeconomic problems. As the infection can progress with mild symptoms that can be misinterpreted as the flu, self-testing methods that can positively identify SARS-CoV-2 are needed to effectively track and prevent the transmission of the virus. In this work, we report a point-of-care toolkit for multiplex molecular diagnosis of SARS-CoV-2 and influenza A and B viruses in saliva samples. Our assay is physically programmed to run a sequence of chemical reactions on a paper substrate and internally generate heat to drive these reactions for an autonomous extraction, purification, and amplification of the viral RNA. Using our assay, we could reliably detect SARS-CoV-2 and influenza viruses at concentrations as low as 50 copies/μL visually from a colorimetric analysis. The capability to autonomously perform a traditionally labor-intensive genetic assay on a disposable platform will enable frequent, on-demand self-testing, a critical need to track and contain this and future outbreaks.

Paper-based multi-well depletion ELISA

“…The paper-based flow controller utilized timer gates that relied on the delamination of the wetted paper from a laminating tape to coordinate different flows. 10,25 The controller was created by imprinting patterns on paper with different types of inks that produced different levels of adhesion between the paper and the laminating tape (Fig. S4A † and Materials and methods).…”

“…S1 †): (1) cellulose filter paper (Whatman grade 1 filter paper) was patterned by drawing lines with water-insoluble ink containing a hydrophobic resin (Sharpie metallic permanent marker, manufacturer part number 2003899). 10,25 The drawn multiple lines form the pattern of 96-or 48-wells where a liquid is impenetrable. The patterned well sheet was then perforated along the vertical lines to aid alignment when folding.…”

“…Instead of serial dilution and manual dispensing of the sample into microwells, our approach relies on gradual dilution of the target analyte as it is depleted from the sample by immobilized capture elements during flow to create a concentration gradient for titer measurement. To automate the sequence and timing of applying different reagents used to perform ELISA, we equipped a lateral flow device with an imprinted flow controller programmed with delaminating timer gates 10,25 that eliminated the need for manual supervision of the assay. To demonstrate practical applications of the developed technology, we created lateral flow devices to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers (both IgG and IgM to spike and nucleocapsid protein) and troponin I, a cardiac biomarker, with each device only requiring the sample and ELISA reagents to be loaded for operation.…”

Influenza A, Influenza B, and SARS-CoV-2 Similarities and Differences – A Focus on Diagnosis

“…Saliva samples analyzed through qRT-PCR confirmed COVID-19 diagnosis in the patients that were used to validate the assay clinically. The multiplex panel was in 100% concordance with PCR testing ( Lee et al, 2021 ). Similarly, Zhang et al also described an RT-LAMP-based method for the multiplex detection of SARS-CoV-2 and Influenza.…”

Paper-Based Biosensors for the Detection of Nucleic Acids from Pathogens

“…Hydrogen ions are generated as a byproduct when a DNA polymerase integrates a dNTP during amplification, which is sufficient to generate a measurable pH change when DNA amplification occurs in a weakly buffered situation [ 96 ]. While the proton release is ubiquitous to all polymerase-based amplification methods, LAMP has been the preferred method for pH-based nucleic acid detection due to its high rate of product formation (>50× PCR yield) within a short period (10–15 min) [ 163 ]. Furthermore, while PCR-based amplification undergoes a background pH shift due to temperature cycling, isothermal amplification maintains a uniform background pH.…”