One-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) for closed-tube colorimetric detection of banana bract mosaic virus in Banana (Musa spp.).

Abstract

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 120µM 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 0.1pg 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.