Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with recombinase polymerase amplification

Abstract

Pasteurella multocida (P. multocida) is an important pathogenic bacterium that poses a serious threat to the development of the livestock economy and human health. Currently, the existing methods for P. multocida detection are time-consuming and require complex professional operations, limiting the application of field detection. In the study, we presented a single-pot naked-eye CRISPR-Cas12a platform (Cas12a-NEye) for the detection of P. multocida. The round tube cover allowed more Cas12a detection solution to be temporarily stored than the flat cap, enabling single-pot assays and avoiding aerosol contamination. The positive samples generated obvious red using naked eye using no excitation light and the negative samples generated blue. The limit of detection (LOD) was a single copy, without cross-reactivity with other closely related bacteria. Furthermore, we validated this platform using 16 P. multocida clinical lung samples and obtained consistent results with the real-time quantitative polymerase chain reaction (qPCR) method. The entire experimental process included rapid DNA extraction (<1 h) and Cas12a-NEye assay (25 min), which was accomplished within 1.5 h. Thus, this “sample-to-answer” platform has significant potential for P. multocida detection.