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

Anal Chim Acta. 2022 Apr 1:1200:339590. doi: 10.1016/j.aca.2022.339590. Epub 2022 Feb 15.

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

MeSH terms

  • COVID-19 Testing
  • COVID-19* / diagnosis
  • Humans
  • Molecular Diagnostic Techniques
  • Nucleic Acid Amplification Techniques / methods
  • Point-of-Care Systems
  • RNA, Viral / analysis
  • RNA, Viral / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Reverse Transcription
  • SARS-CoV-2* / genetics
  • Sensitivity and Specificity

Substances

  • RNA, Viral

Supplementary concepts

  • LAMP assay