1. Rapid and sensitive detection of Dasheen mosaic virus infecting elephant foot yam by reverse transcription loop mediated isothermal amplification of coat protein gene
- Author
-
S. Kamala and T. Makeshkumar
- Subjects
Gel electrophoresis ,Electrophoresis ,Time Factors ,biology ,Potyvirus ,Loop-mediated isothermal amplification ,Temperature ,India ,Reverse Transcription ,biology.organism_classification ,Virology ,Molecular biology ,Sensitivity and Specificity ,Virus ,Reverse transcriptase ,Amorphophallus ,Capsid Proteins ,Pathogen ,Gene ,Reverse Transcription Loop-mediated Isothermal Amplification ,Nucleic Acid Amplification Techniques ,Plant Diseases - Abstract
Dasheen mosaic virus (DsMV), the pathogen causing mosaic disease of elephant foot yam (Amorphophallus paeoniifoilius) is disseminated mainly through vegetative propagation of the tubers. For the rapid and sensitive detection of the virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay based on the coat protein gene has been developed. A final concentration of 5.4 mM magnesium sulphate and 0.7 M betaine in the reaction mixture was found to be optimum for getting characteristic ladder like bands of the amplified product after gel electrophoresis. The reaction was set at 65°C for 50 min followed by reaction termination at 86°C for 5 min in a water bath. The sensitivity of the assay was found to be 100 times higher than that of RT-PCR. The virus was indexed successfully from tubers of elephant foot yam. In tube detection of the DsMV was carried out using fluorescence detection reagents. The assay was validated with field samples from various regions of Kerala state, India.
- Published
- 2014