Exploration of the optimum reaction conditions for the reverse transcription of mycoviral RNA from Lentinula edodes.

From the paper

Abstract

Reverse transcription (RT) of viral RNA to complementary DNA (cDNA) is essential for mycovirus detection; however, the stable secondary structures of double-stranded RNA (dsRNA) and the genomic diversity of viruses complicate this process. This study systematically optimized RT conditions for 15 mycoviruses infecting Lentinula edodes using RT-qPCR, focusing on thermal denaturation temperatures (no heat denaturation [CK], 65 °C [T65], 95 °C [T95], and 98 °C [T98]) and dimethyl sulfoxide (DMSO) concentrations (0-20 %). For purified LeV-HKB dsRNA, T95 and T98 increased cDNA yields by over 50,000- and 100,000-fold, respectively, compared to T65. In total RNA extracts from infected L. edodes, T98 increased cDNA yields for dsRNA mycoviruses by approximately 5-fold compared to CK. Conversely, T95/T98 did not improve cDNA yields for ssRNA viruses, and even resulted in slightly reduced yields for certain viruses. DMSO exerted virus-specific and temperature-dependent effects on RT efficiency: moderate concentrations (5-15 %) enhanced cDNA yields at CK or T65, whereas high concentrations (e.g., 20 %) reduced yields at T95 or T98. No universal DMSO concentration was optimal across viruses, necessitating tailored approaches. Overall, T98 is recommended for dsRNA viruses, CK/T65 for ssRNA viruses, and T98 as a practical compromise for mixed RNA types. This protocol improves detection sensitivity and reliability, thereby facilitating diagnosis and research on fungal RNA viruses.