Development of rapid visual detection technology for Nosema bombycis based on CRISPR/Cas12a system

Silkworm is an important economic insect in China, but very vulnerable to infection by a variety of pathogens in the process of breeding. Among them, the parasitism ofNosema bombycis can cause pebrine disease of silkworm (PDS), seriously endanger the production of silkworm seeds, and lead to huge economic losses. Timely detection of N. bombycis is an effective measure to prevent the outbreak of PDS in the early stage of infection, so a rapid and accurate detection technology suitable for on-site detection of N. bombycis is very important. In order to break through the application limita-tions of the existing detection technology for N. bombycis, in this study, combining Recombinase Polymerase Amplification (RPA) technology and CRISPR/Cas12a system, by optimizing the key components concentration, we successfully estab-lished a rapid visual detection technology for N. bombycis including CRISPR/Cas12a fluorescence detection and CRISPR/ Cas12a immunochromatographic detection. Compared with the traditional nucleic acid detection technology, the detection sensitivity of this technology was up to 2 copies/mu L, the detection limit of N. bombycis genome was 2 fg/mu L, and can detect as low as 100 microsporidia added in 100 healthy silkworm eggs, showing high sensitivity. By detecting silkworm tissues infected with different pathogens, we demonstrated that CRISPR/Cas12a detection technology has good specificity. The accuracy rate of the actual sample is 100%. The technology has the advantage of rapidness, accuracy, visualization, low cost, low equipment requirements and simple operation, and is suitable for the on-site detection of N. bombycis.