CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei

Abstract

<i>Enterocytozoon hepatopenaei</i> (EHP) is a parasite that infects pacific whiteleg shrimp, <i>Penaeus vannamei</i>, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting <i>ptp2</i> gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.