Colorimetric detection platform for banana bunchy top virus (BBTV) based on closed-tube loop mediated isothermal amplification (LAMP) assay

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.

Duke Scholars

Author Smita K Nair Surgery, Surgical Sciences