Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated...

Singleplex and multiplex real-time (TaqMan®) RT-PCR assays were developed to detect seven fig-infecting viruses, i.e., fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV), fig mosaic virus (FMV), fig latent virus 1 (FLV-1), fig cryptic virus 1 (FCV-1) and fig fleck-associated virus (FFkaV). The sensitivity of the newly developed TaqMan® assays was compared with the corresponding conventional RT-PCR (RT-PCR) using 10° to 10^-6 serial dilutions of both cDNA and crude fig extracts. The results showed that the Taqman® RT-PCR assays were generally 10² to 10³-fold more sensitive than the RT-PCR assays, except in the case of FLV-1 detection, where the two techniques had the same sensitivity. In the multiplex Taqman® RT-PCR, only a maximum of five viruses could be detected simultaneously in naturally infected fig trees, regardless of which combination of the virus-specific probes and primers were used. Both the RT-PCR and Taqman® RT-PCR assays were used in a large-scale survey of 100 field-grown fig trees in Egypt. The results showed the presence of all seven viruses under study, mostly occurring as mixed infections (63 %). The prevalence of infections observed in the tested samples were as follows: FMV (62 %), FFkaV (59 %), FLMaV-2 (32 %), FLV-1 (16 %), FLMaV-1 (14 %), FCV-1 (7%) and FMMaV (4%). FMV was invariably associated with diseased trees that presented mosaic-like symptoms. In the few cases where the mosaic-affected trees were found to be free of FMV, they were found to be infected with a mixture of two or more other viruses.