Tersus polymerase

Mouse anti-fluorescein antibodies (anti-FAM) were purchased from Bialexa (Moscow, Russia). Oligonucleotides with modifications (5-carboxyfluorescein (FAM), biotin, 5-carboxyrhodamine-X [ROX], and BHQ2) were synthesized by Syntol (Moscow, Russia). DNAseI, EnGene LbCas12a, T7 RNA polymerase, RNAse inhibitor, NTP, Bst 2.0 WarmStart polymerase, an RNA cleanup kit, and a Monarch DNA gel extraction kit were purchased from NEB (Ipswich, MA, USA). Unmodified oligonucleotides, dNTP, Tersus polymerase, 100+ bp (100–1500 bp) DNA ladder, and M2 means 1 kb (250–10,000 bp) DNA ladder were obtained from Evrogen (Moscow, Russia). Protein A was produced by Imtek (Moscow, Russia). HAuCl₄ and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The membranes as components of lateral flow strips were purchased from Advanced Microdevices (Ambala Cantt, India). All salts and organic compounds had analytical-grade purity.

Kits for Nfo-RPA were manufactured by TwisDx (Maidenhead, UK). Unlabeled primers were synthesized by Evrogen. Biotin- and FAM-labeled primers were synthesized by Syntol (Moscow, Russia). Kits of total DNA/RNA extraction from plants and an RNAse inhibitor (RNAsin) were purchased from Syntol. A FAM-labeled THF-containing oligonucleotide probe was synthesized by BioResearch (Risskov, Denmark). The mix for PCR contained SYBR Green I, dNTP, Tersus polymerase, and Moloney murine leukemia virus (MMLV) revertase, and the kits for DNA extraction from gels were purchased from Evrogen (Moscow, Russia). T7 RNA polymerase, DNAse I, RNA cleanup kit, and NTPs were purchased from Neb (Ipswich, MA, USA). Mouse monoclonal IgG (clone 2A3c) specific to fluorescein (anti-FAM) was produced by Bialexa (Moscow, Russia). Recombinant streptavidin and goat anti-mouse IgG were produced by Imtek (Moscow, Russia). Ethylenediaminetetraacetic acid (EDTA), HAuCl₄, bovine serum albumin (BSA), and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Salts, buffers, organic solvents, and other compounds were analytical grade.
For LFT-producing nitrocellulose membrane CNPC12, PT R5 fiberglass membrane, sample pad membrane GFB-R4, and absorbent pad AP045 were purchased from Advanced Microdevices (Ambala Cantt, India).

All original plasmid constructs are accessible through the Addgene plasmid repository (https://www.addgene.org). The fADL-1e-HA-linker-p3 (Addgene ID: 139440) and fADL-1e-flag-linker-p3 (Addgene ID: 139441) vectors are based on phage fADL-1e vector (Antibody Design Laboratories, San Diego, CA, USA) with the addition of linker sequence that was fused with the p3 protein of fd filamentous bacteriophage and encodes an HA-tag (hemagglutinin tag) or 3xFLAG for detection, serine-glycine linkers for conformational delimitation, and NcoI and NheI sites for the cloning of the desired peptide sequences. We used the following peptides: P#1—CILDLPKFC—positive ligand for Raji-FL cells; P#2—HKLIHAASERVLSDARTILEENIQDQDVLLLIKKRAPSPLPKMA—irrelevant protein; P#3—PTCNIRVTVCSFDDGVDLPP—irrelevant protein; and P#4—PKYVKQNTLKLAT—positive control substrate for HLA-DR1 peptide fragment of influenza A virus hemagglutinin [35 (link)]. PCR was performed on a Thermal cycler T100 (BioRad, Berkeley, CA, USA) using Tersus polymerase (Evrogen, Moscow, Russia).