posted on 2021-02-04, 20:03authored bySoo Khim Chan, Pinyi Du, Caroline Ignacio, Sanjay Mehta, Isabel G. Newton, Nicole F. Steinmetz
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.