High-Throughput Sequencing-Guided Development and Validation of an RT-dPCR Assay for Sensitive Detection of Lentinula edodes Spherical Virus in Shiitake Mushrooms.

Shiitake (Lentinula edodes) cultivation is often affected by virus-associated degeneration, necessitating sensitive and quantitative diagnostics. This study used high-throughput sequencing (HTS) as a preliminary screening approach to identify the major mycoviruses present in symptomatic samples and to guide target selection for assay development. HTS identified Lentinula edodes spherical virus (LeSV) together with other shiitake-associated mycoviruses, and LeSV was selected as the target for assay development. Based on these results, a nanoplate-based reverse transcription digital PCR (RT-ndPCR) assay was developed for LeSV. The assay demonstrated high analytical specificity, with no cross-reactivity with LeNSRV1 or LeV, and achieved approximately 10-fold higher detection than RT-qPCR. When applied to 30 field samples, RT-ndPCR detected LeSV in 100% (30/30) of samples compared with 86.7% (26/30) by RT-qPCR, identifying additional low-titer positives. These findings demonstrate that RT-ndPCR provides sensitive detection and absolute quantification of LeSV for shiitake surveillance and health certification.