High Sensitivity, Rapid Detection of Virus in High Traffic Environments

Abstract

The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.

Carbon and Graphene Quantum Dots as Bionanomaterials: A Perspective View of COVID–19

“…As commercially available antiviral drugs to treat the COVID-19 disease are limited and most of the drug's approval was based on emergency used authorization (EUA), it is essential to have a rapid and highly sensitive detection of the virus to control the pandemic (Waller et al, 2022). For the transmission of the virus, the spike (S) protein (Figure 2: Left) of the respective virus is the key glycoprotein for the entry of the virus into the host cells (Wang and Xiang, 2020).…”

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

“…Colorimetry is particularly attractive in developing POCT for untrained users; for example, many at-home rapid tests utilize paper-based LFAs with a colorimetric readout. Colorimetric readout is most often achieved using a pH indicator [ 64 , 104 , 105 ] or nanoparticle aggregation [ 105 , 106 , 107 ]. Assays leveraging colorimetric readout typically include multiple separate reactions performed in parallel, enabling de facto multiplex detection.…”

“…During amplification, a pH decrease is caused by the production of protons as more nucleic acids are produced. Within a weakly buffered solution, pH-sensitive dyes, such as phenol red, will change color as the pH shifts during amplification [ 105 ]. Acid-base color readouts are time sensitive and do not exhibit a color change indefinitely, which may be caused by nucleic acid degradation [ 108 ]; the color change must be observed within 30 to 60 min to remain sensitive, which could pose a restriction.…”

“…In an interesting example, Waller et al developed LAMP assays that use both pH-based and LFA-based colorimetric readouts in a single workflow for high-traffic testing of SARS-CoV-2 ( Figure 5 a) [ 105 ]. The LAMP assay can be achieved in a 96-well plate for high throughput, and the binary readout can be analyzed quickly by a camera in an automated process before further testing the positive wells for specific genes on the LFA [ 105 ]. Using logical negative controls, multiplex detection can be implemented with only two colors in a separate reaction.…”

“…LFAs often use strong biotin and streptavidin binding interaction for detection mechanisms that can be applied with appropriate labeled antigens/antibodies and AuNPs. As shown in Figure 6 d, dye-coated, streptavidin-conjugated polymeric nanoparticles flow through and bind to the biotin; through the NP aggregations, visible multiplexed detections can be read [ 105 , 107 ]. In another example utilizing nucleic acid amplification, RPA primers labeled with biotin on the 5′ end and digoxigenin (hapten) on the 3′ can also bind with neutravidin-conjugated carbon NPs on specific regions on a lateral flow test demonstrated by Cherkaoui et al, highlighted in Figure 6 e [ 69 ].…”