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

A batch processed titanium–vanadium oxide nanocomposite based solid-state electrochemical sensor for zeptomolar nucleic acid detection

“…The experiments also demonstrated that the sensing response of 20%-TVO and the SPE was analogous to each other in detecting clinically relevant 100 copies (2.5 copies per mL) and the presence of human genomic DNA or serum did not hinder the sensing response of TVO. As demonstrated in our other study 44 concerning the magnetocapture assay optimization, the sample-toanswer time is 2 h that includes 25 min for magneto-extraction, 80 min (1 h for amplication, and 20 min for inactivation) for LAMP, and 10 min for electrochemical measurement. The amplication time can further be reduced to 1 h if the reaction is terminated at an earlier time, making the nal sample-toanswer time total 1 h. Both are signicantly shorter than the sample-to-answer time for real-time PCR (total of 2.5 h, 1 h for nucleic acid extraction, and 1.5 h for PCR).…”

“…The experiments concerning the LAMP primer optimization were conducted as described in a separate study from our group. 44 Briey, the LAMP assay involved 0.4 mM outer primers, 0.332 mM forward and backward inner primers, 1 mM forward loop primers, and 0.4 mM back loop primers in their nal concentration 45 (sequences, F3: CGA TAA GTA TGT CCG CAA TT, B3: GCT TCA GAC ATA AAA ACA TTG T, FIP: ATG CGT AAA ACT CAT TCA CAA AGT CCA ACA CAG ACT TTA TGA GTG TC, BIP: TGA TAC TCT CTG ACG ATG CTG TTT AAA GTT CTTTAT GCT AGC CAC, loop F: TGT GTC AAC ATC TCT ATT TCT ATA G, and loop B: TCA ATA GCA CTT ATG CAT CTC AAG G). A 25 mL LAMP assay also contained the following components in their respective nal concentrations: 1Â Bst 2.0 DNA polymerase reaction buffer [20 mM Tris-HCl, 50 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Tween-20, and pH 8.8], dNTPs (1.4 mM), SYBR I (1Â diluted from 10 000Â stock), 8 U of Bst 2.0 DNA polymerase, MgSO 4 (6 mM), template (plasmid #14567, https://www.addgene.org/145671/ or in vitro transcribed RNA, 1 mL) or magneto-extracted nucleic acid on beads (2 mL).…”

“…The experiments involving the in vitro transcription of SARS-CoV-2 RdRp RNA from plasmid and its concentration assessment were performed as described in a separate study from our group. 44 Briey, the RdRp sequence bearing plasmid (#14567, https:// www.addgene.org/145671/) was linearized using the restriction enzyme SnaBI. The linearized plasmid was then treated with an in vitro transcription kit, resulting in the generation of RdRp RNA.…”

“…The working scheme of the indirect magnetocapture (optimized in a separate paper from our group 44 ) has been illustrated in Fig. 8.…”

“…It should be noted that the SPE and real-time uorescence measurement data in this manuscript are independent of our other manuscript focusing on magnetocapture optimization. 44 In the current study, end-point SWV measurements were carried out aer electrochemical in situ LAMP was performed on magneto-extracted SARS-CoV-2 plasmid and RNA. The grouped column bar graph for the peak current (shown in Fig.…”