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. 2021 Sep;32(3):148-157.
doi: 10.7171/jbt.21-3203-010.

Rapid, Affordable, and Scalable SARS-CoV-2 Detection From Saliva

Andrew Hayden  1 , Marcy Kuentzel  1 , Sridar V Chittur  1   2
Affiliations

Rapid, Affordable, and Scalable SARS-CoV-2 Detection From Saliva

Andrew Hayden et al. J Biomol Tech. 2021 Sep.

Abstract

Here we present an inexpensive, rapid, and robust reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection method that is easily scalable, enabling point-of-care facilities and clinical labs to determine results from patients' saliva directly in 30 minutes for less than $2 per reaction. The method uses a novel combination of widely available reagents that can be prepared in bulk, plated, and frozen and remain stable until samples are received. This innovation dramatically reduces preparation time, enabling high-throughput automation and testing with time to results (including setup) in less than 1 hour for 96 patient samples simultaneously when using a 384-well format. By using a dual reporter (phenol red pH indicator for end-point detection and SYTO-9 fluorescent dye for real time), the assay also provides internal validation of results and redundancy in the event of an instrument malfunction.

Keywords: Fluorescent LAMP; RT-LAMP; extraction-free detection; saliva detection.

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Figures

FIGURE 1
FIGURE 1
Schematic of dual-assay workflow. RT, room temperature; FIP, forward inner primer; BIP, backward inner primer, F3, forward outer primer; B3, backward outer primer; LB, loop backward primer.
FIGURE 2
FIGURE 2
Primer optimization.
FIGURE 3
FIGURE 3
Sensitivity and specificity testing. MM, master mix.
FIGURE 4
FIGURE 4
Comparative performance metrics of polyvinylsulfonic acid (PVSA) vs. RNaseOUT. MM, master mix.
FIGURE 5
FIGURE 5
Sensitivity and specificity testing with polyvinylsulfonic acid (PVSA). MM, master mix; ST, standard.
FIGURE 6
FIGURE 6
Stability testing. MM, master mix.
See this image and copyright information in PMC

References

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