Abstract
Nucleic acid amplification tests (NAATs) such as quantitative real-time reverse transcriptase PCR (qRT-PCR) or isothermal NAATs (iNAATs) such as loop-mediated isothermal amplification (LAMP) require pure nucleic acid free of any polymerase inhibitors as its substrate. This in turn, warrants the use of spin-column mediated extraction with centralized high-speed centrifuges. Additionally, the utilization of centralized real-time fluorescence readout and TaqMan-like molecular probes in qRT-PCR and real-time LAMP add cost and restrict their deployment. To circumvent these disadvantages, we report a novel sample-to-answer workflow comprising an indirect sequence-specific magneto-extraction (also referred to as magnetocapture, magneto-preconcentration, or magneto-enrichment) for detecting SARS-CoV-2 nucleic acid. It was followed by in situ fluorescence or electrochemical LAMP. After in silico validation of the approach’s sequence selectivity against SARS-CoV-2 variants of concern, the comparative performance of indirect and direct magnetocapture in detecting SARS-CoV-2 nucleic acid in the presence of excess host nucleic acid or serum was probed. After proven superior, the sensitivity of the indirect sequence-specific magnetocapture in conjunction with electrochemical LAMP was investigated. In each case, its sensitivity was assessed through the detection of clinically relevant 10 2 and 10 3 copies of target nucleic acid. Overall, a highly specific nucleic acid detection method was established that can be accommodated for either centralized real-time SYBR-based fluorescence LAMP or portable electrochemical LAMP.
Keywords: Electrochemical LAMP; Magnetic sequence-specific target enrichment; Quantitative real-time LAMP; SARS-CoV-2.
【저자키워드】 SARS-CoV-2., Quantitative real-time LAMP, Magnetic sequence-specific target enrichment, Electrochemical LAMP, 【초록키워드】 SARS-CoV-2, qRT-PCR, SARS-CoV-2 variant, in silico, amplification, nucleic acid, serum, sensitivity, PCR, Nucleic acid detection, NAAT, isothermal amplification, molecular, inhibitor, Quantitative, Disadvantages, isothermal, sequence, SARS-CoV-2 nucleic acid, polymerase, probe, Host, approach, real-time reverse transcriptase, investigated, clinically, restrict, circumvent, turn, 【제목키워드】 SARS-CoV-2 nucleic acid,