Development of rapid multiplex human herpesvirus detection systems based on recombinase polymerase amplification and...

Human herpesviruses (HHVs) are highly prevalent globally (e.g., Epstein-Barr virus with over 95% prevalence) and can persist latently for life, causing severe conditions like blindness, encephalitis, and even lethal infections in neonates. HHV reactivation is of particular clinical concern in transplantation medicine, where immunosuppression can trigger viral reactivation, leading to graft rejection, secondary infections, and increased mortality. Notably, latently infected cells show no pathological changes, their contamination in cell cultures not only undermines the accuracy of scientific data and reliability of research results but also risks public health through the transmission of biological products. This study constructed three detection systems by combining multiplex Polymerase Chain Reaction (PCR) with Recombinase Polymerase Amplification (RPA) and Lateral Flow Assay (LFA): the Octaplex PCR system, with a sensitivity of 1 × 10⁴ copies per 50 μL system, suitable for high-throughput contamination screening of cell samples (1 out of 30 tested samples was EBV positive); the Quadruplex RPA system, which improved sensitivity to 1 × 10³ copies per 50 μL system (10 times higher than the PCR system), and shortened reaction time to 15-20 min, applicable for rapid preliminary screening; And the Duplex RPA-LFA detection system, maintaining 1 × 10³ copies per 50 μL system sensitivity, enabling visual result detection within 20 min without complex instruments for on-site rapid identification. These systems provide reliable tools for cell quality control, technical support for early diagnosis of HHVs-associated diseases and prevention strategies, as well as theoretical references for other virus detection systems, boasting broad application prospects.