Development of a diagnostic protocol for Cucumber mosaic virus for screening banana ( Musa spp.) planting material in Ivory Coast.

摘要

Cucumber mosaic virus (CMV) is the primary virus affecting commercial banana (Musa spp.) plantations in Ivory Coast, with the disease also present within nursery systems. However, currently no virus indexing of planting material is performed for banana in the country. This current study attempts to quantify levels of detection of CMV within banana material using flinders technology associates (FTA) cards in order to identify a reliable and reproducible method suitable for local conditions. A suite of PCR primers were developed against CMV coat protein open reading frame (CP ORF) sequence available on GenBank. TaqManReg. assay generated comparable CT values with either FTA or cetyltrimethyl ammonium bromide (CTAB) derived templates and was able to detect CMV RNA in leaf samples. The conventional real time polymerase chain reaction (RT-PCR) assay, RNeasy and FTA derived RNA templates reliably amplified CMV, whilst CTAB-derived RNA gave variable results. The TaqManReg. reaction was much more sensitive than the conventional RT-PCR for CMV detection in banana leaf samples. The suitability of FTA cards as a matrix for virus nucleic acid capture provides scope for its use in the transportation of banana virus samples to laboratories for diagnosis and characterisation, circumventing constraints of time-related degeneration of sample integrity. Sequence data of the CMV CP ORF region of 16 isolates from Cote d'Ivoire clustered the isolates to CMV Sub-Group IA. This is the first successful application of Real-time PCR and FTA technology for a banana virus in Africa.