Development of multiplex RT-PCR for the detection of two main mycoviruses infecting Chinese Lentinula edodes germplasm resource

Abstract:

[Background] Lentinula edodes virus disease is important for L. edodes production. Previous to this study, L. edodes mycovirus HKB (LeV-HKB) and L. edodes partitivirus 1 (LePV1) were proved to be the two major mycoviruses identified in L. edodes germplasm, and co-infection of these two viruses was also prevalent. [Objective] The purpose of this study was to develop a quick and efficient technique for detection of these two viruses in the early stage of L. edodes production. [Methods] Three pairs of specific primers for LeV-HKB, LePV1 and actin genes (β-actin) of L. edodes were designed based on the reported sequences, respectively. The crucial factors of multiplex RT-PCR including template concentration, primers concentration, dNTPs concentration, annealing temperature, and number of cycles were then optimized. [Results] A multiplex RT-PCR detection method for LeV-HKB (or LeV-HKB related virus) and LePV1 was developed. [Conclusion] The multiplex RT-PCR assay developed in this study had high specificity and repeatability using the virus detection of L. edodes core collections.