Journal: Biosensors

Article Title: A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System

doi: 10.3390/bios15120807

Figure Lengend Snippet: Specificity of WNV DETECTR. ( A , B ) Viral RNAs from WPV1a (lentivirus pseudotyped with WNV1a capsid gene), WPV2 (lentivirus pseudotyped with WNV2 non-coding sequence between membrane and envelope genes), CPV (lentivirus pseudotyped without any WNV genes) (1.0 × 10 4 CFU), and from SEOV 80-39, IAV, IBV, JEV, DENV-2, and ZIKV (1.0 × 10 4 PFU) were extracted using Punch-it™ NA-Sample Kit (Nanohelix, Daejeon, Republic of Korea) and amplified by RT-RPA. RT-RPA products were analyzed by 2-Step WNV DETECTR with fluorescence readout. Fluorescence saturation was measured within 10 min after incubation of the LbaCas12a-gRNA complex with the RT-RPA products at 37 °C. Error bars represent mean ± s.e. ( n = 3 replicates; * p < 0.05 between 0 min and 10 min samples, two-sample t -test). CFU, colony-forming unit; PFU, plaque-forming unit; WPV1a, WNV1a pseudovirus; WPV2, WNV2 pseudovirus; CPV, control pseudovirus; NTC, non-template control.

Article Snippet: Both the WNV1a capsid gene (nucleotide 156 to 256) (GenBank accession number: KY703854 ) and the WNV2 non-coding sequence between the membrane and envelope genes (nucleotide 1462 to 1636) (GenBank accession number: AY532665.1 ) [ ] were synthesized (Macrogen, Seoul, Republic of Korea) and amplified by PCR using Phusion ® High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs, Ipswich, MA, USA) with specific primers containing a T7 promoter for each lineage ( ).

Techniques: Sequencing, Membrane, Amplification, Fluorescence, Incubation, Control