Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1

Human bocavirus (HBoV) 1 is considered an important pathogen that mainly affects infants aged 6-24 months, but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging. Herein, we present a novel faster, lower cost, reliable method for the detection of HBoV1, which integrates a recombinase polymerase amplification (RPA) assay with the CRISPR/Cas12a system, designated the RPA-Cas12a-fluorescence assay. The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37°C without the need for sophisticated instruments. The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens. Furthermore, the method was appraised using 28 clinical samples, and displayed high accuracy with positive and negative predictive agreement of 90.9% and 100%, respectively. Therefore, our proposed rapid and sensitive HBoV1 detection method, the RPA-Cas12a-fluorescence assay, shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care. The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection. The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.