A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test

Sci Rep. 2021 Nov 1;11(1):21385. doi: 10.1038/s41598-021-00827-0.

Abstract

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 / epidemiology*
  • COVID-19 / virology
  • COVID-19 Nucleic Acid Testing / methods*
  • Genome, Viral
  • Humans
  • Infection Control / methods
  • Limit of Detection
  • Mass Screening / methods
  • Molecular Diagnostic Techniques / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • Pandemics*
  • RNA, Viral / genetics
  • RNA, Viral / isolation & purification
  • RNA-Directed DNA Polymerase / genetics
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcription / genetics
  • SARS-CoV-2 / genetics*
  • Sensitivity and Specificity

Substances

  • RNA, Viral
  • RNA-Directed DNA Polymerase

Supplementary concepts

  • LAMP assay