One-Step RT-LAMP: A Method for Closed-Tube Colorimetric Detection of RNA Viruses in Plants

A good virus indexing platform is essential for early detection of plant viruses and for providing virus free planting materials. Majority of the plant viruses are RNA viruses and the available detection methods include ELISA and RT-PCR. The ELISA is time consuming, and RT-PCR needs post PCR sample handling, predisposing to sample cross contamination. The LAMP (loop-mediated isothermal amplification) is an auto-cycling strand displacement isothermal DNA synthesis catalyzed by a strand displacing DNA polymerase like the Bst DNA polymerase. The RT-LAMP (reverse transcription LAMP) is a specific, highly sensitive, and relatively fast diagnostic method for RNA viruses. In one-step RT-LAMP, the total RNA extracted from symptomatic samples can be directly amplified under isothermal conditions by incorporating a reverse transcriptase and an RNase inhibitor in the LAMP assay. The inclusion of hydroxy naphthol blue dye in the RT-LAMP reaction mixture facilitates closed-tube visual detection of positive samples by a color change from violet to sky blue. Molecular fidelity of the LAMP amplicons can be assessed through restriction profiling.