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

Abstract

Abstract 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.

Keywords

• CRISPR‐Cas12a
• detection
• human bocavirus 1
• on‐site diagnosis
• recombinase polymerase amplification