One‐Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point‐of‐Care Testing

ABSTRACT Hepatitis E virus (HEV) poses a serious threat to both public health and animal food safety, thereby highlighting the demands for rapid, sensitive, and easy‐to‐use detection. This study aimed to develop a One‐Pot assay using CRISPR/Cas13a for detecting HEV RNA, suitable for point‐of‐care testing (POCT) in resource‐limited settings. CRISPR/Cas13a combined with reverse transcription polymerase chain reaction (RT‐PCR) and reverse transcription recombinase‐aided amplification (RT‐RAA) was applied to a One‐Pot assay device. Additionally, a large cohort of HEV‐infected patient (154) and animal (104) specimens was utilized for validation. The RT‐PCR/RT‐RAA + CRISPR/Cas13a assays for HEV RNA detection (genotypes: HEV‐1, HEV‐3, and HEV‐4) were established, optimized, and validated, achieving a limit of detection (LoD) of 1 copy/μL and 100% specificity. In the application validation for HEV infection, the positive rates of the RT‐PCR + CRISPR and RT‐RAA + CRISPR assays were 98.6% and 89.6% for patients, and 96.6% and 88.8% for animals, respectively, which were superior to those of RT‐qPCR. Furthermore, sample rapid lysis, reagent lyophilization, and the One‐Pot device were integrated to construct a One‐Pot assay with an LoD of 10 2 copies/μL. Despite slight decreases in sensitivity, the One‐Pot assay significantly reduces the assay time to 35 min, making it easy to perform, minimizing contamination, and meeting the requirements for screening. We developed a One‐Pot assay of HEV RNA using the CRISPR/Cas13a which effectively realizes a POCT test and maximizes the impetus for POCT implementation and shows potential as a valuable tool for detecting and monitoring HEV infection.