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.
【초록키워드】 SARS-CoV-2, coronavirus, protocol, reliability, Transcription, virus, RNAs, RNA, RT-LAMP, RT-qPCR, Region, stability, Internal control, nucleocapsid, Control, target, reverse transcription, isothermal amplification, Quantitative, VLP, isothermal, COVID-19 patient, plant virus, Consensus, acute respiratory syndrome, Virus-like particle, gold standard, RT-qPCR assays, RNase P, clinical setting, positive control, reagent, plasmids, molecular assays, VLPs, bacteriophage Qβ, encapsidation, polymerase chain, shown, lack, include, assays, required, overcome, highlighting, internal and external, the SARS-CoV-2, 【제목키워드】 COVID-19, Particle, RT-LAMP, Control,