Table 1.
RPA primers and gRNAs for SFTSV DETECTR.
Fig 1.
(A) Graphical workflow of SFTSV DETECTR. (B) Schematic presentation of positive and negative results obtained using a lateral flow strip. In a positive result, a band may appear at the C-line possibly due to the partial cleavage of the FB-reporter by Cas12a. FQ-reporter, fluorescence-quencher reporter; FB-reporter, FAM-Biotin lateral flow strip reporter; C-line, control line; T-line, test line.
Fig 2.
Detection of lentiviruses pseudotyped with SFTSV S gene using DETECTR combined with fluorescence assay.
(A) A diagram of gRNAs and primer sets for SFTSV DETECTR. (B and C) Lentiviruses pseudotyped with and without SFTSV S gene (SPV and CPV, respectively) (100 CFU), IAV and IBV (100 PFU) and HCV and HEV (100 genome copies) were lysed via HUDSON and viral nucleic acids amplified via RT-RPA with a primer set specific for the SFTSV S gene. RT-RPA amplicons were detected with SFTSV DETECTR combined with fluorescence assay using gRNAs (A) S1 and (B) S2 corresponding to the S genes of all SFTSV genotypes. Fluorescence saturation occurred within 10 min. Values are presented as means ± s.d. (error bars) (n = 3 replicates; * p < 0.05 between samples, two-sample t-test). nt, nucleotides; PFU, plaque forming unit; CFU, colony forming unit; SPV, SFTSV pseudovirus; CPV, control pseudovirus.
Fig 3.
Different concentration of lentiviruses pseudotyped with SFTSV S gene (1.0 × 10−1 to 1.0 × 102 CFU per reaction) were lysed through HUDSON and viral nucleic acids amplified via RT-RPA with a primer set specific for the SFTSV S gene. RT-RPA amplicons were detected using SFTSV DETECTR combined with fluorescence assay or lateral flow using gRNAs (A) S1 and (B) S2 corresponding to the S genes of all SFTSV genotypes. Fluorescence saturation occurred within 10 min. Lateral flow assay results were evaluated 2 min after the strip was reacted with the sample. Values are presented as means ± s.d. (error bars) (n = 3 replicates; * p < 0.05 between samples, two-sample t-test). C-line, control line; T-line, test line; NTC, no template control. CFU, colony forming unit.
Fig 4.
Application of SFTSV DETECTR to clinical samples.
(A) RNA was extracted from patient plasma samples and subjected to RT-PCR for SFTSV detection. PCR products (124 bp) were visualized using gel electrophoresis. (B, C) HUDSON-treated patient plasma samples were used to amplify viral nucleic acids using RT-RPA with a primer set specific for the SFTSV S gene. RT-RPA amplicons were detected using SFTSV DETECTR combined with fluorescence assay or lateral flow using gRNAs (A) S1 and (B) S2 corresponding to the S genes of all SFTSV genotypes. Fluorescence saturation occurred within 10 min. Lateral flow assay results were evaluated 2 min after the strip was reacted with the sample. Values are presented as means ± s.d. (error bars) (n = 3 replicates; * p < 0.05 between samples, two-sample t-test). C-line, control line; T-line, test line; NTC, no template control.