Multiplex Detection of Pleurotus ostreatus Mycoviruses

Abstract

Mycoviruses are viruses that specifically infect and replicate in fungi. Several mycoviruses have been previously reported in Pleurotus ostreatus, including the oyster mushroom spherical virus (OMSV), oyster mushroom isometric virus (OMIV), Pleurotus ostreatus spherical virus (POSV), and Pleurotus ostreatus virus 1 (PoV1). This study was designed to develop a multiplex RT-PCR for simultaneous detection and differentiation of the four P. ostreatus mycoviruses. Four pairs of primers were designed from conserved regions based on the reported sequences and the multiplex RT-PCR products were 672 bp for OMSV, 540 bp for OMIV, 310 bp for POSV, and 200 bp for PoV1. The optimal annealing temperature of the multiplex RT-PCR was 62 °C and the detection limits of the plasmids were 100 fg for OMSV and OMIV and 1 pg for POSV and PoV1. This technique was successfully applied for the detection of OMSV, OMIV, and POSV from different P. ostreatus strains and the plasmid containing the PoV1 sequence. This methodology can serve as a powerful diagnostic tool for the survey of the incidence and epidemiology of the four P. ostreatus mycoviruses, further contributing to the prevention and treatment of mycoviral diseases in P. ostreatus.

Oyster Mushroom Spherical Virus Crosses the Species Barrier and Is Pathogenic to a New Host Pleurotus pulmonarius

“…PCR cycling was performed as follows: 95 °C for 3 min, then 34 cycles of amplification with 95 °C for 30 s, 53 °C for 50 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. For multiplex PCR, the program was set as follows: 95 °C for 5 min, followed by 30 cycles of 3 steps including 95 °C for 30 s, 62 °C for 30 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min [ 48 ]. Amplification products were electrophoresed at 125 V for 40 min in 1% agarose gel.…”