Evaluation of indirect sequence-specific magneto-extraction-aided LAMP for fluorescence and electrochemical...

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 10² and 10³ 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.