Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B

Figures

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  Fig. 1. Schematic diagrams of RT-RPA-Cas12a-mediated fluorescence assay for the detection of human rhinovirus B (HRV-B). (A) Sequence alignment of available twenty whole genomes of HRV-B. The conserved nucleotide sequence in each position are presented by asterisks. Coloring of the nucleotide sequence was made according to the same pattern presented in MEGA7 software. (B) A schematic illustration different crRNA spacer sequence sites within the target VP4 gene sequence of HRV-B species genome. crRNA spacer sequences are indicated by blue colored text, respectively. RT-RPA: reverse-transcription recombinase polymerase amplification assay

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  Fig. 2. Screening of the optimal RT-RPA primers using agarose gel electrophoresis. M, DL2000 DNA marker; 1–6, RT-RPA products were amplified using the combination of different primers, including HRV-B-F1/HRV-B-R1, HRV-B-F1/HRV-B-R2, HRV-B-F2/HRV-B-R1, HRV-B-F3/HRV-B-R2, HRV-B-F3/HRV-B-R3, and HRV-B-F3/HRV-B-R4, respectively

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  Fig. 3. Evaluation the performance of different crRNA sequence in RT-RPA-Cas12a-mediated fluorescence assay with ssDNA FQ. (A) Representative real-time fluorescence kinetics of single crRNA sequence in the RT-RPA-Cas12a-mediated fluorescence assay. (B) Fluorescence values of single crRNA sequence obtained at 15–25 min. The data were shown as mean ± SD in each group with at least three independent experiments. NC, negative control group

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  Fig. 4. Specificity evaluation of RT-RPA-Cas12a-mediated fluorescence assay using RNA from viral samples, including human rhinoviruses B (HRV-B) species, the mixture of human rhinoviruses A and C species (HRV-AC) in equal volume, metapneumovirus (HmPV), adenovirus (ADV), bocavirus (HBOV), and respiratory syncytial virus (RSV), as the template of RT-RPA, respectively. (A) Specificity evaluation of RT-RPA-Cas12a-mediated real-time fluorescence assay with a fluorescence detector readout. (B) Specificity examination of the RPA-Cas12a-mediated end-point fluorescence assay at 15 min. The data are presented as mean ± SD (n = 3). NC, negative control group

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  Fig. 5. Examination of the sensitivity of the RT-RPA-Cas12a-mediated fluorescence assay using a serial dilution of the standard RNA of human rhinovirus B (HRV-B) as the template. (A) Sensitivity examination of RT-RPA-Cas12a-mediated real-time fluorescence assay for HRV-B. (B) Sensitivity evaluation of RT-RPA-Cas12a-mediated end-point fluorescence assay at different times. The values were presented as the means ± SD in each group with at least three independent experiments. NC, negative control group