Reverse transcription loop-mediated isothermal amplification assay for rapid and sensitive detection of Banana bract mosaic virus in cardamom (Elettaria cardamomum)

Abstract

Cardamom being a perennial and propagated vegetatively, Banana bract mosaic virus (BBrMV) in cardamom spreads mainly through infected material. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for easy and quick detection of the virus. The following conditions proved optimal for amplification: 4 mM of magnesium sulphate, 1.2Mof betaine, 65 °C, and 1 h of reaction time. The results were assessed visually by turbidity and green fluorescence (induced by adding manganese chloride and calcein) in the reaction tube and also by gel electrophoresis. The assay successfully detected the virus in infected plants whereas no crossreaction was recorded with healthy plants. The detection limit for RT-LAMP was up to 100 times that for conventional RT-PCR and on a par with that for realtime RT-PCR. The assay was validated by testing field samples of cardamom plants from different cardamomgrowing tracts in Kerala, India.