RESUMO
The banana bract mosaic virus (BBrMV) is a major virus affecting bananas and plantains. Banana being propagated vegetatively, there arises a high risk of virus transmission through planting materials. Available molecular detection technique like the Reverse Transcriptase Polymerase Chain Reaction needs post-amplification sample handling, predisposing to sample cross contamination. A one-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) assay coupled with colorimetric detection was optimised for easy and quick detection of BBrMV in banana. The viral coat protein gene was amplified under isothermal conditions at 65 ºC. The RT-LAMP assay was optimised with respect to concentrations of MgSO4, dNTP, Bst polymerase enzyme and HNB dye. The total RNA purified from symptomatic samples was directly amplified under isothermal conditions by including 100 U M-MLV reverse transcriptase and 20 U RNasin® plus RNase inhibitor in the reaction. With the addition of 120 µM of Hydroxy Naphthol Blue (HNB) dye in the RT-LAMP reaction, the BBrMV-positive samples had a colour change from violet to sky blue after the reaction. The RT-LAMP assay detected BBrMV in 0.1 pg of total RNA isolated from symptomatic plants. Molecular characterisation of RT-LAMP products was done using restriction profiling and sequence analysis. The RT-LAMP assay was validated using field-collected banana leaf samples. The assay successfully detected the virus from symptomatic samples while the healthy samples showed no amplification. Samples sourced from banana plants with symptoms of banana bunchy top virus, banana streak virus and cucumber mosaic virus tested negative in the RT-LAMP assay, thus ensuring the specificity of the assay.
RESUMO
A colorimetric closed-tube Loop mediated isothermal amplification (LAMP) assay was developed for rapid and sensitive detection of banana bunchy top virus (BBTV) from leaf and sucker tissues of infected banana plants. Six LAMP primers were designed targeting BBTV coat protein gene. Isothermal amplification was set at 65 °C and end point detection made by including hydroxy naphthol blue dye in the reaction where the positive samples showed colour change from violet to sky blue. Molecular characterization of LAMP amplicon was made with restriction digestion and sequencing. Restriction digestion with Sau3AI having single cut site within the LAMP internal primer flanking region yielded two fragments of expected size. Sequence analysis confirmed that amplification corresponded to BBTV coat protein gene. Comparison of LAMP assay with conventional PCR showed that LAMP assay was 1000 times more sensitive than conventional PCR in BBTV detection with a detection limit of 0.05 ng total DNA per reaction. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00784-w.
