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

Les Jones, Abhijeet Bakre, Hemant Naikare, Ravindra Kolhe, Susan Sanchez, Yung-Yi C Mosley, Ralph A Tripp

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)e0257563
JournalPloS one
Volume16
Issue number9
DOIs
StatePublished - 2021

Keywords

  • 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

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