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

• Published in: European journal of plant pathology 2014-02, Vol.138 (2), p.209-214
• Main Authors: Siljo, A, Bhat, A. I
• Format: Article
• Language: English
• Subjects: Agriculture; Banana bract mosaic virus; betaine; Biomedical and Life Sciences; cardamom; cross reaction; detection limit; Ecology; Elettaria cardamomum; field experimentation; fluorescence; gel electrophoresis; Herbs; Life Sciences; Magnesium sulfate; Manganese; Plant diseases; Plant Pathology; Plant Sciences; Polymerase chain reaction; reverse transcriptase polymerase chain reaction; reverse transcription; Turbidity; viruses
• ISSN: 0929-1873; 1573-8469
• DOI: 10.1007/s10658-013-0318-0

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, 1.2 M of 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 cross-reaction 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 real-time RT-PCR. The assay was validated by testing field samples of cardamom plants from different cardamom-growing tracts in Kerala, India.