Journal Article Research Support, Non-U.S. Gov't Epublish 14 citations
PubMed: 34534259 DOI: 10.1371/journal.pone.0257563
Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs.
PLoS One 16(9):e0257563
Authors: Les Jones, Abhijeet Bakre, Hemant Naikare, Ravindra Kolhe, Susan Sanchez, Yung-Yi C Mosley, Ralph A Tripp
Affiliations
- Department of Infectious Disease, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America.
- State of Georgia COVID-19 Taskforce, Athens, Georgia, United States of America.
- Tifton Diagnostic and Investigational Laboratory, University of Georgia, Athens, Georgia, United States of America.
- Medical College of Georgia, Augusta University, Augusta, Georgia, United States of America.
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.
MeSH
Rna, Viruses, Sars Virus, Reverse Transcription, Covid-19, Sars-Cov-2
Cited by
- Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives. Chem Sci, 2023
- Rapid Detection of SARS-CoV-2 RNA in Human Nasopharyngeal Specimens Using Surface-Enhanced Raman Spectroscopy and Deep Learning Algorithms. ACS Sens, 2023
- Point-of-care isothermal nucleic acid amplification tests: progress and bottlenecks for extraction-free sample collection and preparation. Expert Rev Mol Diagn, 2024
- Diagnostic assay and technology advancement for detecting SARS-CoV-2 infections causing the COVID-19 pandemic. Anal Bioanal Chem, 2022
- The Complexity of SARS-CoV-2 Infection and the COVID-19 Pandemic. Front Microbiol, 2022
- Isothermal amplification using sequence-specific fluorescence detection of SARS coronavirus 2 and variants in nasal swabs. Biotechniques, 2022