Deoxynucleoside triphosphates dntps

Escherichia coli BL21 strains and the pET28a expression vector were purchased from Novagen. The pBT and pTRG vectors, E. coli XR reporter strains, and reagents for the one-hybrid assay were obtained from Stratagene. All enzymes including DNA polymerase, restriction enzymes and DNA ligase, deoxynucleoside triphosphates (dNTPs), and all antibiotics were purchased from TaKaRa Biotech. PCR primers were synthesized by Invitrogen. All plasmids constructed for the purposes of our study are listed in Table S1.

The selected 28 isolates were further characterised based on their cell morphology, Gram-reaction, KOH method, sporulation, and catalase and API 50 CHL assays (BioMérieux, Marcy-l’Étoile, France) [55 (link)]. Genotypic identification was performed by sequencing the 16S rRNA gene. The reaction mixture for the amplification step of the 16S rRNA region contained 1× GoTaq Flexi buffer without MgCl₂ (Applied Biosystems, Foster City, CA, USA), 1.5 mM MgCl₂ (Applied Biosystems, Foster City, CA, USA), 0.025 U/µL Taq polymerase (TaKaRa, Ohtsu, Japan), 1.5 µM primers UNI16SF (5′-GAGAGTTTGATCCTGGC-3′) and UNI16SR (5′-AGG AGG TGA TCC AGC CG-3′) [56 (link)], 0.2 mM deoxynucleoside triphosphates (dNTPs) (TaKaRa, Ohtsu, Japan) and 1 ng/µL DNA. The PCR programme consisted of an initial denaturation at 96 °C for 5 min, 30 cycles of 30 s at 96 °C, 30 s at 55 °C and 30 s at 72 °C, followed by 5 min of final elongation at 72 °C. The resulting PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s recommendations and sequenced at Macrogen (Amsterdam, The Netherlands). The 16S rRNA sequences were aligned with the NCBI nt database, using the BLASTn algorithm v. 2.11.0 [57 (link)] to find nucleotide sequence homologues (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 18 February 2020).

All oligonucleotides, including the ASFV specific target sequence, primers, crRNAs (designed using benchling), reporters, and lateral flow capture probes were synthesized by Sangon Biotech (Shanghai, China) (Table 1). Biotin-labeled goat anti-mouse IgG (H+L) dispensed on the control line was from Beyotime Biotechnology (Shanghai, China). LbaCas12a, NEB buffer 3.1, Bst 2.0 WarmStart polymerase was purchased from New England Biolabs (Ipswich, MA, USA). Deoxynucleoside triphosphates (dNTPs) and Taq polymerase premix were purchased from Takara (Beijing, China).
Gold trichloride acid (HAuCl₄.3H₂O), trisodium citrate, and streptavidin (SA) were purchased from Sigma–Aldrich (Steinheim, Germany). Absorbent and fiberglass papers and nitrocellulose membranes were purchased from Sartorius AG (Gottingen, Germany). A dispenser to immobilize the biotin-labeled goat anti-mouse IgG and probe DNA on the LFB and a nitrocellulose membrane cutter were purchased from Shanghai Kinbio (Shanghai, China). A portable strip reader was from Goldbio Technology Co. (Shanghai, China). Genome and plasmid extraction kits were purchased from Qiagen (Hilden, Germany) and Tiangen Biotech (Beijing, China), respectively. All buffers used in this study were prepared in our laboratory. Other chemicals were purchased from standard commercial sources and were of pure analytical grade.