[One-Step Detection of Human Influenza B Virus Through Recombinase Polymerase Amplification and CRISPR/Cas12a Protein]

Abstract

Objective: To establish a one-step detection method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a protein for the rapid and sensitive detection of human influenza B virus.

Methods: RPA amplification primers were designed according to the conserved gene (NS1 gene) of human influenza B virus (Victoria lineage). The reaction system was established using the standard plasmid as the template. First of all, the reaction system was incubated at 37 ℃ for 15 minutes for RPA amplification. Then, the CRISPR/Cas12a system on the tube cap was thoroughly mixed with the RPA amplification product at the bottom of the tube through fast centrifugation, and real-time fluorescence detection was carried out at 37 ℃. The reaction conditions were optimized to establish a one-step RPA-CRISPR/Cas12a detection method for human influenza B virus. The sensitivity of the testing method was evaluated using standard plasmids and pseudoviruses, and the specificity was evaluated using other viruses that may cause febrile respiratory syndrome. The consistency between the results of the one-step detection method and those of RT-qPCR detection was evaluated by testing real samples.

Results: A one-step detection method based on RPA-CRISPR/Cas12a was successfully established. The optimal reaction conditions included a reaction temperature of 37 ℃, a Cas12a/crRNA concentation ratio of 1∶1, a Cas12a concentration of 120 nmol/L, a single-stranded DNA (ssDNA) probe concentration of 300 nmol/L, and a primer concentration of 480 nmol/L. The method could detect standard plasmid DNA as low as 2.8 copies/μL within 25 minutes and pseudoviruses as low as 2.77 copies/μL within 30 minutes. The testing method showed high specificity, and no cross-reaction was observed with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A (H1N1) virus, or respiratory syncytial virus subgroup A. When testing clinical samples, the sensitivity and the specificity for examining clinical samples were 93.33% and 100%, respectively, and consistency with RT-qPCR results was 97.14%.

Conclusion: With the one-step detection method based on RPA-CRISPR/Cas12a established in this study, the whole sample detection process, including nucleic acid release, reverse transcription, isothermal amplification, CRISPR/Cas12a system cleavage, and fluorescence signal output, can be completed within 30 minutes. Its high sensitivity, specificity, and successful application in clinical samples highlight its potential for rapid point-of-care testing in clinical settings.

Keywords: CRISPR-Cas systems; Fluorimetry; Human influenza B virus; Recombinase polymerase amplification.

图 1

Workflow and schematic diagram of RPA-CRISPR/Cas12a one-step assay RPA-CRISPR/Cas12a一步法流程及原理图 SSB: single-stranded binding; ssDNA: single-stranded DNA; dsDNA: double-stranded DNA; crRNA: CRISPR-derived RNA; PAM: protospacer adjacent motif. A, Workflow; B, principle of detection.

图 2

Primer screening 引物筛选 A, Screening of RPA primers for NS1 gene of human influenza B virus (lane 1: 250 bp DNA ladder; lane 2/3: F1R1 NTC group/amplification group; lane 4/5: F2R2 NTC group/amplification group; lane 6/7: F3R3 NTC group/amplification group. The concentration of the NS1 gene plasmid of influenza B virus was 10⁵ copies/μL). B, Preliminary exploration of the sensitivity of RPA amplification with F2R2 primer set (lane 1: 250 bp DNA ladder; lane 2: NTC; Lane 3: 10¹ copies/μL; lane 4: 10² copies/μL; lane 5: 10³ copies/μL; lane 6: 10⁴ copies/μL; lane 7: 10⁵ copies/μL). NTC: amplification negative control, amplification was performed using DEPC-treated water instead of plasmid.

图 3

Optimization of reaction conditions 反应条件优化 NTC: no template control for amplification in which amplification was performed using DEPC-treated water instead of plasmid. A, Reaction temperature; B, Cas12a/crRNA ratio; C, Cas12a concentration; D, ssDNA probe concentration; E, primer concentration. The concentration of plasmid was 10⁴ copies/μL. The error lines represent the standard deviation of triplicate experiments. ^** P < 0.01, ^*** P < 0.001.

图 4

Analysis of sensitivity and specificity 灵敏度分析和特异性分析 NTC: no template control for amplification in which amplification was performed using DEPC-treated water instead of plasmid. A, Sensitivity analysis using plasmid as the template; B, sensitivity analysis using pseudoviruses as templates; C, specificity analysis of using pseudoviruses as templates. The error lines represent the standard deviation of triplicate experiments. ^*** P < 0.001, vs. NTC group.