Abstract
Mycoviruses are viruses that naturally infect and replicate in fungi. They are widespread in all major fungal groups including plant and animal pathogenic fungi. Several dsRNA mycoviruses have been reported in Aspergillus fumigatus. Multiplex polymerase chain reaction (PCR) amplification is a version of PCR that enables amplification of different targets simultaneously. This technique has been widely used for detection and differentiation of viruses especially plant viruses such as those which infect tobacco, potato and garlic. For rapid detection, multiplex RT-PCR was developed to screen new isolates for the presence of A. fumigatus mycoviruses. Aspergillus fumigatus chrysovirus (AfuCV), Aspergillus fumigatus partitivirus (AfuPV-1), and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) dsRNAs were amplified in separate reactions using a mixture of multiplex primer pairs. It was demonstrated that in the presence of a single infection, primer pair mixtures only amplify the corresponding single virus infection. Mixed infections using dual or triple combinations of dsRNA viruses were also amplified simultaneously using multiplex RT-PCR. Up until now, methods for the rapid detection of Aspergillus mycoviruses have been restricted to small scale dsRNA extraction approaches which are laborious and for large numbers of samples not as sensitive as RT-PCR. The multiplex RT-PCR assay developed here will be useful for studies on determining the incidence of A. fumigatus mycoviruses. This is the first report on multiplex detection of A. fumigatus mycoviruses.
Highlights
IntroductionAspergillus species (spp.) are universal and ubiquitous in nature, and are usually found as saprophytes in soil, decaying vegetation, seed and grains [1]
Aspergillus is a genus in the phylum Ascomycota, kingdom Fungi
A. fumigatus strains used in this study were isolates A56, A88 and Af293 for Aspergillus fumigatus chrysovirus (AfuCV), Aspergillus fumigatus partitivirus-1 (AfuPV-1) and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1), respectively [5]
Summary
Aspergillus species (spp.) are universal and ubiquitous in nature, and are usually found as saprophytes in soil, decaying vegetation, seed and grains [1]. They are important both medically and commercially. Research on dsRNA elements showed that various Aspergillus spp. were infected by mycoviruses [4] (Varga et al., 1998). Different dsRNA mycoviruses have been reported in A. fumigatus with an 18.6% incidence in Holland [6].
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