Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays
Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays
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DOI：
10.1021/acs.biomac.0c01727
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发表时间：
2021-02-04
影响因子：
6.2
通讯作者：
Steinmetz, Nicole F.
中科院分区：
文献类型：
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作者：
Chan, Soo Khim;Du, Pinyi;Steinmetz, Nicole F.
逆转录环介导等温扩增(RT-LAMP)是检测SARS冠状病毒2型(SARS-CoV-2)的金标准逆转录定量聚合酶链式反应(RT-qPCR)的一种快速、廉价的等温方法。然而，与RT-qPCR不同的是，没有一致的检测区域或最佳的RT-LAMP方法，而且大多数协议不包括内部对照以确保可靠性。来自传染性新冠肺炎患者的裸RNA、质粒，甚至是RNA已被用作RT-LAMP检测的外部阳性对照，但此类试剂缺乏全程对照所需的稳定性。为了克服缺乏合适的内外阳性对照和检测RNA的不稳定性，我们利用噬菌体Qβ和植物病毒豌豆褪绿斑驳病毒(CCMV)构建了包裹靶RNA的病毒样颗粒(VLP)，即所谓的SARS-CoV-2 LAMP检测模块(SLDM)。目的RNA为SARS-CoV-2核衣壳(N)基因和人RNaseP基因(内对照)的截短片段，作为RT-qPCR和RT-LAMP的阳性对照。稳定地包裹在Qβ和CCMV VLP中的靶RNA先前被证明在RT-qPCR检测中起到全程对照的作用，这里我们证明SLDM可以完成RT-LAMP和拭子到检测(直接RT-LAMP热裂解)检测的相同功能。SLDM在临床环境中得到了验证，突出了VLP作为分子分析阳性对照的前景。
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 Q beta 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 Q beta 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.