Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneou s detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mo saic virus and Cucumber mosaic virus, which have ssRNA genomes. The detecti on of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneou sly from a mixed infection. Identification and detection of individual viru ses was achieved through the visualisation of discretely sized PCR amplicon s by gel electrophoresis. Alternatively, a colourimetric microplate detecti on system utilising digoxigenin-labelled virus-specific probes was used. Th e latter assay was up to five times more sensitive than detection by gel el ectrophoresis and between 25 and 625 times more sensitive than ELISA for th e various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental i nteractions between heterologous templates and primers. (C) 2000 Elsevier S cience B.V. All rights reserved.