A Comparative Evaluation of Indirect Sequence-Specific Magnetoextraction-aided Fluorescence and Electrochemical LAMP with SARS-CoV-2 Nucleic Acid as the Analyte

Since the beginning of the SARS-CoV-2 pandemic, nucleic acid amplification test (NAAT) such as quantitative real-time reverse transcriptase PCR (qRT-PCR) has remained the primary intervention for diagnostics and containment of SARS-CoV-2. Despite its remarkable clinical as well as analytical specificity and sensitivity, qRT-PCR necessitates pure nucleic acid free of any polymerase inhibitors (from complex biological matrices) as its substrate. Similarly, isothermal NAATs (iNAATs), despite their advantage over qRT-PCR in terms of thermal cycler independence, still require pure nucleic acid as a template. The requirement of pure nucleic acid in turn warrants the use of spin-column mediated extraction with centralized high-speed centrifuges. Additionally, utilization of centralized real-time fluorescence readout and use of sequence-specific molecular probes like TaqMan further prevent their deployment in decentralized locations. To circumvent these disadvantages, we have envisioned a sample-to-answer workflow comprising of indirect sequence-specific magneto-extraction (also referred to as magnetocapture, magneto-preconcentration, or magneto-enrichment in this manuscript) followed by in situ fluorescence or electrochemical LAMP. This study, using SARS-CoV-2 nucleic acid as the analyte, compared the analytical effectiveness of indirect and direct sequence-specific magneto-extraction followed by LAMP. Since contamination carryover may affect the efficacy of sequence-specific indirect magnetocapture, its performance in presence of excess host nucleic acid or serum was probed. Through these experiments, we have established a comprehensive limited resource-adoptable and highly specific nucleic acid detection method with the limit of detection of 2.5 copies/L. Its advantage lies in the flexibility of using either centralized real-time SYBR-based fluorescence LAMP or portable electrochemical LAMP as the readout. Simultaneously, the performance with magneto-capture aided fluorescence and electrochemical LAMP readouts were weighed against each other in terms of analytical sensitivity, specificity, and turnaround time. Additionally, the analytical efficacy of the magnetocapture-LAMP workflow was also checked against that of LAMP using pure nucleic acid as a template. Besides being the first report utilizing electrochemical LAMP to detect SARS-CoV-2 nucleic acid, this would probably be the first study to make the analytical comparative assessment of magnetic preconcentration combined with in situ fluorescence and electrochemical LAMP. It is probably also the first study (to the best of our knowledge) to compare the analytical efficacy of a sequence-specific magnetic target enrichment-LAMP (fluorescence and electrochemical) to that of a LAMP assay using pure nucleic acid as a template.