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

Abstract

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.

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“…In a separate manuscript from our groups currently undergoing peer review (uploaded with this manuscript 40 ), we have demonstrated the utility of the indirect magnetocapture over the direct magnetocapture in terms of assay performance. The working scheme of the indirect magnetocapture, the one utilized in this manuscript has been illustrated in Figure 8.…”

“…The experiments concerning the LAMP primer optimization and assays have been described in detail in a separate manuscript from our groups currently undergoing peer review (uploaded with this manuscript 40 ). Briefly, the LAMP assay involved 0.4 μM outer primers, 0.332 μM forward and backward inner primer, 1 μM forward loop primers and 0.4 μM back loop primers in their final concentration 41 were conducted in real-time PCR (64C for 1 h, with fluorescence monitoring every 1 min, followed by melting curve analysis) or thermal cycler (64C for 1 h followed by 85C for 20 min), respectively.…”

“…The experiments involving the in vitro transcription of SARS-CoV-2 RdRp RNA from plasmid and its concentration assessment is described in detail in a separate manuscript from our groups currently undergoing peer review (uploaded with this manuscript 40 ). Briefly, the RdRp sequence bearing plasmid (#14567, https://www.addgene.org/145671/) was linearized using the restriction enzyme SnaBI.…”

“…Electrochemical read-out using fabricated TVO electrode After characterization and optimization, the 20%-TVO electrode was used for electrochemical detection of SARS-CoV-2 RdRp plasmid DNA and in vitro transcribed RNA in an aqueous solution using isothermal amplification (LAMP). The LAMP primer optimization, in vitro transcription, and RNA concentration determination has been described briefly in the materials and methods section and in more detail in a separate manuscript focusing on the magnetocapture aspect of the project (currently undergoing peer review, uploaded with this manuscript 40 ). Figure 6a schematically represents (1) the LAMP amplification, (2) the tools used for detecting the amplification, and (3) the sensing principle.…”

“…Limit of detection (LOD) study: (uploaded with this manuscript 40 ). The sensing response for SWV measurement employing the 20%-TVO electrode was then compared with that of a commercially purchased screen-printed electrode (SPE) and real-time fluorescence (Figure 7).…”