CRISPR/Cas13a-mediated visual detection: A rapid and robust method for early detection of Nosema bombycis in silkworms

Abstract

The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/μL, 10 copies/μL, and 1 copy/μL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/μL, 10 fg/μL, and 1 fg/μL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings.