Elsevier

Analytica Chimica Acta

Volume 1200, 1 April 2022, 339590
Analytica Chimica Acta

A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR

https://doi.org/10.1016/j.aca.2022.339590Get rights and content

Highlights

  • The direct RT-LAMP assay developed is simple and extraction-free, requiring only heating and bead-assisted mechanical lysis for sample preparation.

  • A calibrated template transfer tool was devised to simplify sample addition into RT-LAMP reactions.

  • The assay is rapid and has a high diagnostic performance of 99.30% accuracy, 98.81% sensitivity, and 100% selectivity compared to RT-PCR.

Abstract

The global public health crisis and economic losses resulting from the current novel coronavirus disease (COVID-19) pandemic have been dire. The most used real-time reverse transcription polymerase chain reaction (RT-PCR) method needs expensive equipment, technical expertise, and a long turnaround time. Therefore, there is a need for a rapid, accurate, and alternative technique of diagnosis that is deployable at resource-poor settings like point-of-care. This study combines heat deactivation and a novel mechanical lysis method by bead beating for quick and simple sample preparation. Then, using an optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to target genes encoding the open reading frame 8 (ORF8), spike and nucleocapsid proteins of the novel coronavirus, SARS-CoV-2. The test results can be read simultaneously in fluorometric and colorimetric readouts within 40 min from sample collection. We also calibrated a template transfer tool to simplify sample addition into LAMP reactions when pipetting skills are needed. Most importantly, validation of the direct RT-LAMP system based on multiplexing primers S1:ORF8 in a ratio (1:0.8) using 143 patients’ nasopharyngeal swab samples showed a diagnostic performance of 99.30% accuracy, with 98.81% sensitivity and 100% selectivity, compared to commercial RT-PCR kits. Since our workflow does not rely on RNA extraction and purification, the time-to-result is two times faster than other workflows with FDA emergency use authorization. Considering all its strengths: speed, simplicity, accuracy and extraction-free, the system can be useful for optimal point-of-care testing of COVID-19.

Keywords

RT-LAMP
Point-of-care
COVID-19
SARS-CoV-2
Rapid testing
Bead beating

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