A one-pot RPA-CRISPR detection method for point-of-care testing of Enterocytozoon hepatopenaei infection in shrimp

The pathogen Enterocytozoon hepatopenaei (EHP) has significantly affected the shrimp farming industry since the last decade. For effective prevention, accurate and reliable detection methods for EHP infection are important, especially the methods suitable for point-of-care testing (POCT) environment. By combining recombinase po-lymerase amplification (RPA) with the CRISPR-Cas12a system, this study developed a POCT-friendly detection method for EHP infection with one-pot procedure. The combined method utilized robust amplification capacity of RPA and high specificity of CRISPR, thus had better accuracy and reliability. The one-pot procedure was achieved by careful design and screening of the crRNA and systematic optimization of the reaction. The issues related to the compatibility of RPA and Cas12a cleavage have been successfully solved. The method was highly specific and the sensitivity reached 101 gene copies per reaction. The practical performance was validated with clinical shrimp samples. With the generous temperature requirement and convenient end-point reading of re-sults, the one-pot method is highly suitable for POCT environment. It is a good example of one-pot RPA-CRISPR detection assay for POCT purpose, and has great application value in prevention of EHP infection in shrimp farming industry.

Automated summary

This study developed a point-of-care testing (POCT)-friendly detection method for Enterocytozoon hepatopenaei (EHP) infection by combining recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system. The method showed improved accuracy and reliability by utilizing the robust amplification capacity of RPA and high specificity of CRISPR. By carefully designing crRNA and optimizing the reaction, the one-pot procedure was achieved, solving the compatibility issues between RPA and Cas12a cleavage. The method demonstrated high specificity and sensitivity, making it suitable for POCT environment and having great application value in preventing EHP infection in the shrimp farming industry.