Evaluation of indirect sequence-specific magneto-extraction-aided LAMP for fluorescence and electrochemical SARS-CoV-2 nucleic acid detection

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 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.

Keywords: Electrochemical LAMP; Magnetic sequence-specific target enrichment; Quantitative real-time LAMP; SARS-CoV-2.

Graphical abstract

Scheme 1

Indirect and direct sequence-specific magnetocapture of target nucleic acid (present with host nucleic acid and polymerase inhibitors from serum) leads to probe-target nucleic complex immobilized on the magnetic bead. Following magnetic decantation wash, the target bound magnetic beads was used for in situ LAMP (or reverse transcriptase LAMP) amplification with fluorescence or electrochemical readout.

Fig. 1

Comparison of indirect and direct sequence-specific magnetocapture of 100 copies (1 fg) and 1000 copies (10 fg) of SARS-CoV-2 RdRp plasmid DNA from aqueous solution (Panel B), and solutions spiked with 1 ng hgDNA (Panel C), or serum (10%, v/v, Panel D) followed by in situ qLAMP. Panel A describes the scheme of in situ qLAMP with magnetocaptured SARS-CoV-2 RdRp plasmid DNA. Target control LAMP experiments were performed with 10³ copies of pure plasmid DNA (without any magnetocapture). NTC assays comprised of magnetocapture experiments that were carried out without any target nucleic acid followed by qLAMP. Error bars represent standard deviation (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 2

Indirect magnetocapture of 100 and 1000 copies of in vitro transcribed SARS-CoV-2 RdRp RNA from aqueous media (Panel B), or aqueous sample spiked with hgDNA (1 ng, Panel C), or serum (5%, v/v, Panel D) followed by in situ qRT-LAMP. Panel A describes the scheme of in situ qRT-LAMP with magnetocaptured SARS-CoV-2 RdRp RNA. Target control qRT-LAMP experiments were performed with 10³ copies of RdRp RNA (without any magnetocapture). NTC assays comprised of magnetocapture experiments that were carried out without any target RNA followed by qRT-LAMP Error bars represent standard deviation (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 3

Electrochemical LAMP studies conducted on pure 10¹–10⁴ copies of SARS-CoV-2 RdRp plasmid DNA (Panel B) or RNA/reaction (Panel C) (without magnetocapture). Panel A describes the mechanism of amplicon-mediated methylene blue sequestration and subsequent reduction of current. NTC assays were conducted without any template nucleic acid addition. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Indirect magnetocapture of 100 and 1000 copies of SARS-CoV-2 RdRp plasmid DNA (Panel B) and RNA (Panel C) from aqueous media, aqueous sample spiked with hgDNA (1 ng), or serum followed by in situ electrochemical endpoint (reverse transcription) LAMP (eLAMP or eRT-LAMP). Panel A describes the scheme of in situ eLAMP with magnetocaptured 100 and 1000 copies of SARS-CoV-2 RdRp plasmid DNA or RNA. Target control (TC) eLAMP or eRT-LAMP experiments were performed with 10³ copies of RdRp DNA or RNA, respectively (without any magnetocapture). NTC assays comprised of magnetocapture experiments that were carried out without any target DNA or RNA followed by eLAMP or eRT-LAMP, respectively. Error bars represent standard deviation (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.