PCR-based diagnostic assays for accurate detection of abaca viruses in the laboratory were developed thru DA-BIOTECH R1406. Three assays in multiplex format were developed for detection of (1) SCMV and BBrMV, (2) ABTV genome segments and (3) BBTV genome segments.

 

For detection of SCMV and BBrMV, a reverse transcription-duplex-PCR (RT-duplex-PCR) assay with specific coamplification of internal control from abaca was developed. Three primers sets were designed to amplify fragments of BBrMV coat protein (743bp), SCMV-Ab coat protein (595bp) and Musa spp. elongation factor 1 (219bp). Specificity of each primer pair was confirmed by cloning and sequencing of the RT-PCR products. The RT-duplex-PCR assay was optimized by varying primer concentration combinations, dNTP concentrations, Mg2+ concentrations, Taq polymerase concentrations, annealing temperature and extension temperature. The developed RT-duplex-PCR protocol has the same level of sensitivity and specificity as that of uniplex PCR. The use of internal control eliminates the risk of obtaining false negative results. Analysis of samples collected from various abaca planting regions in the Philippines with the use of the optimized RT-duplex-PCR proves that it is reliable, robust and faster than uniplex PCR detection of each virus. The RT-duplex-PCR assay described is rapid, specific and sensitive for the detection of BBrMV and SCMV-Ab from nucleic extracts of abaca.

 

For detection of ABTV and BBTV, separate multiplex PCR assays were developed for detection of the individual virus’ genome segments. All genome segments were targeted due to the documented negative effect on diagnostic sensitivity of PCR by the viruses’ multicomponent nature. The multiplex PCR methods were optimized at 56.5°C annealing temperature, and was evaluated altogether to have 91-100% diagnostic sensitivity and diagnostic specificity (CI=95%, n=60) against ABTV and BBTV, more accurate than LAMP and uniplex PCR. These assays provide strategic diagnostic tools to circumvent difficulties in diagnosis of multicomponent abaca viruses.