Development and preliminary evaluation of real-time fluorescence-based RPA and RPA-CRISPR/Cas12a assays for rapid...

Coxiella burnetii (C. burnetii) is a ubiquitous zoonotic pathogen that exists widely in nature and is an obligate intracellular parasite of eukaryotic cells, causing Q fever in both humans and animals. Therefore, the surveillance and prevention of Q fever are of great importance for public health and the livestock industry. In this study, specific RPA primers, probes, and crRNAs were designed and synthesized targeting the IS1111 gene of C. burnetii, a real-time fluorescence recombinase polymerase amplification (qRPA) assay and a RPA combined with CRISPR/Cas12a (RPA-CRISPR/Cas12a) assay for the rapid detection of C. burnetii were developed. The qRPA assay completed target gene amplification within 20 min, and the amplification curves were monitored in real time on a portable Genie III instrument at 39 °C. The entire single-tube RPA-CRISPR/Cas12a assay, including RPA amplification and CRISPR/Cas12a detection, was completed within 40 min, and the results could be directly visualized under blue-light illumination. Both assays exhibited high specificity for C. burnetii and showed no cross-reactivity with the tested microorganisms. The LOD of the qRPA assay for C. burnetii DNA standard was 54 copies/μL, whereas the LOD of the RPA-CRISPR/Cas12a assay was 1.3 copies/μL. The assays were further evaluated on 456 clinical samples for the detection of C. burnetii. The postive rates of the qRPA and RPA-CRISPR/Cas12a assays were 3.73% (17/456) and 4.17% (19/456), respectively. Compared with qPCR detection results, the concordance rates were 99.34% and 99.78%, respectively. In conclusion, the qRPA and RPA-CRISPR/Cas12a assays developed in this study are rapid, specific and sensitive, making them suitable for the on-site detection of C. burnetii at primary level.