Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

PLoS One. 2021 Sep 17;16(9):e0257563. doi: 10.1371/journal.pone.0257563. eCollection 2021.

Abstract

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 Nucleic Acid Testing / methods*
  • Diagnostic Tests, Routine / methods*
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • RNA, Viral / isolation & purification
  • Real-Time Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / isolation & purification*
  • Sensitivity and Specificity

Substances

  • RNA, Viral

Supplementary concepts

  • LAMP assay

Grants and funding

Funding was through the Georgia COVID-19 Task Force and the Georgia Research Alliance to RT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.