Multiplex reverse transcription-polymerase chain reaction for simultaneous detection of banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV) in abaca

The abaca (Musa textilis Nee) is an important fibre crop in the Philippines. Its fibre yield and quality is severely impacted by two potyviruses, namely, banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV). A multiplex RT-PCR assay was developed for the simultaneous detection of these two RNA viruses in abaca. Primer sets were designed to amplify fragments of BBrMV coat protein (743 bp), SCMV coat protein (595 bp) and Musa sp. elongation factor 1 (219 bp) as an internal control. The specificity of each primer pair was confirmed by cloning and sequencing of the RT-PCR products. The multiplex RT-PCR assay was optimized by varying primer concentration combinations, dNTP concentration, Mg2+ concentration, Taq polymerase concentration, annealing temperature and extension temperature. The developed multiplex RT-PCR protocol has the same level of sensitivity and specificity when compared with uniplex RT-PCR detection for each virus and internal control. The inclusion of internal control amplification eliminates the risk of obtaining false negative results. The use of the optimized multiplex RT-PCR in simultaneous detection of BBrMV and SCMV from field samples proves that it is reliable, robust and faster than uniplex RT-PCR detection of each virus. This method will be useful for large-scale surveys of these two RNA viruses in abaca and will be vital for epidemiological studies and disease prevention.