Sensitive and Rapid Dual One‐Tube Recombinase Aided PCR Assays for the Simultaneous Detection of Cytomegalovirus, Epstein‐Barr Virus, Human Herpesvirus 6, and Herpes Simplex Virus 1/2 in Whole Blood From Children Undergoing Hematopoietic Stem Cell Transplantation

Early, rapid, and sensitive detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and herpes simplex virus Types 1/2 (HSV-1/2) is critical for improving clinical outcomes in pediatric hematopoietic stem cell transplantation (HSCT). A dual one-tube recombinase-aided PCR (DO-RAP) assay was developed for the simultaneous detection of these viruses. The analytical sensitivity and specificity of the assay were evaluated and subsequently validated using 70 clinical whole blood samples. The DO-RAP assay demonstrated limits of detection (LOD) of two copies/reaction for CMV and HHV-6, 7 copies/reaction for EBV, and three copies/reaction for HSV-1/2. The total detection time was 62 min. In whole blood samples, DO-RAP demonstrated 100% sensitivity relative to qPCR, and identified 37 additional positive cases among qPCR-negative samples. DO-RAP exhibited higher sensitivity than qPCR for monitoring viral status before HSCT, at 0-30 days post-HSCT, and at 31-60 days post-HSCT, enabling earlier detection of CMV, EBV, HHV-6, and HSV-1/2 in recipients. During donor screening, DO-RAP also identified one additional CMV-positive and EBV-positive case compared with qPCR. The DO-RAP assay enables rapid and accurate detection of low-level viral infections in whole blood samples from HSCT settings, providing a valuable tool for routine monitoring of HSCT-associated viral infections.