Field-deployable, rapid diagnostic testing of saliva for SARS-CoV-2

Abstract

To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per µl of saliva and a sensitivity and specificity with clinical samples of > 96%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

“…S20 ). This result directly contradicts literature reports where RT-LAMP assays in saliva utilize RNase inhibitors improve the LoD ( Janíková et al, 2021 ; Wei et al, 2021b ); this discrepancy may be due to the type of RNase inhibitor used. Both Guanidine HCl and carrier DNA improved the LoD ( Fig.…”

“…Dilution reduces the buffering capacity of saliva and decreases the concentration of inhibitory components, both of which would delay colorimetric reporting. We found dilution preferable to more commonly used pre-treatment steps found in a variety of studies, such as pre-treatment with proteases ( Janíková et al, 2021 ; Wei et al, 2021b ), Chelex® 100 ( Yaren et al, 2021 ) or RNA extraction steps to inactivate inhibitory components of saliva ( Garneret et al, 2021 ; Yamazaki et al, 2021 ), as it is less complex on the end-user.…”

“…S18 and Fig. S19 ) and was added to our RT-LAMP reaction formulation for colorimetric solution reactions ( Lalli et al, 2021 ; Wei et al, 2021b ). We did not include these components in our paper RT-LAMP formulation, however, as they resulted in a color change when drying on paper (due to unexplained reasons).…”

“…Finally, uracil-DNA glycosylase (UDG) and deoxyuridine triphosphate (dUTP; Fig. S22 ) were included to reduce carryover contamination ( Hsieh et al, 2014 ; Tang et al, 2016 ; Wei et al, 2021b ; Yaren et al, 2017 ; Zeng et al, 2020 ). When including Guanidine HCl, carrier DNA, and UDG, the LoD of our optimized RT-LAMP colorimetric assay in 5% processed saliva in solution improves to 250 copies/reaction ( Fig.…”

CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout

“…Parallelly, there are serological approaches are in place to detect the contagion, but the process is time-consuming, cost-effective, and unreliable. However, considering the growing need for even more extensive testing, the fact that RT-PCR requires advanced infrastructure and up to 24 h to deliver the results, warrants a better alternative (Lübke et al 2020 ; Wei et al 2021 ). Therefore, scientists have developed CRISPR (clustered regularly interspaced palindromic repeats)-based easy-to-use kits for detecting SARS-CoV-2 from the contagion samples in 30–60 min, just like a pregnancy test which does not require any sophisticated instruments or well-established lab and can be used in the home, airports, and even in railway stations for faster and efficient detection of contagion compared to WHO (World Health Organization)/CDC (Centers for Disease Control and Prevention) approved RT-PCR method (Surjit et al 2006 ; Hou et al 2020 ).…”