Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays

Biomacromolecules. 2021 Mar 8;22(3):1231-1243. doi: 10.1021/acs.biomac.0c01727. Epub 2021 Feb 4.

Abstract

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and inexpensive isothermal alternative to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, unlike RT-qPCR, there are no consensus detection regions or optimal RT-LAMP methods, and most protocols do not include internal controls to ensure reliability. Naked RNAs, plasmids, or even RNA from infectious COVID-19 patients have been used as external positive controls for RT-LAMP assays, but such reagents lack the stability required for full-process control. To overcome the lack of proper internal and external positive controls and the instability of the detection RNA, we developed virus-like particles (VLPs) using bacteriophage Qβ and plant virus cowpea chlorotic mottle virus (CCMV) for the encapsidation of target RNA, namely a so-called SARS-CoV-2 LAMP detection module (SLDM). The target RNA is a truncated segment of the SARS-CoV-2 nucleocapsid (N) gene and human RNase P gene (internal control) as positive controls for RT-qPCR and RT-LAMP. Target RNAs stably encapsidated in Qβ and CCMV VLPs were previously shown to function as full-process controls in RT-qPCR assays, and here we show that SLDMs can fulfill the same function for RT-LAMP and swab-to-test (direct RT-LAMP with heat lysis) assays. The SLDM was validated in a clinical setting, highlighting the promise of VLPs as positive controls for molecular assays.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bromovirus* / chemistry
  • Bromovirus* / genetics
  • COVID-19 Nucleic Acid Testing / standards*
  • COVID-19* / diagnosis
  • COVID-19* / genetics
  • Humans
  • Molecular Diagnostic Techniques / standards*
  • Nucleic Acid Amplification Techniques / standards*
  • SARS-CoV-2 / genetics*

Supplementary concepts

  • LAMP assay