Rapid and visual detection of Lentinula edodes spherical virus using CRISPR/Cas12a-based RT-RPA

Lentinula edodes (shiitake) is a globally important edible mushroom, but viral infections can hinder mycelial growth, reduce yield, and degrade quality, leading to economic losses. This study examined viral infections in L. edodes cultivated in South Korea. We developed a CRISPR/Cas12a-based reverse transcription recombinase polymerase amplification (RT-RPA) assay for the rapid and accurate detection of L. edodes spherical virus (LeSV), the predominant mycovirus-infecting L. edodes in Korea. The optimized RT-RPA-CRISPR/Cas12a assay can be completed within 20 min at 34 °C and demonstrated 100 times higher sensitivity than RT-PCR. Field sample verification further confirmed its superior detection rate over RT-PCR. The RT-RPA-CRISPR/Cas12a method described herein has the potential to facilitate early diagnosis of LeSV infections and support disease management in L. edodes cultivation.