Fast and reliable detection of Plum pox virus in woody host plants using the Blue LAMP protocol

Up to now, the polymerase chain reaction is the most widely used method for the amplification of nucleic acids in vitro, especially for pathogen detection because of its high sensitivity. In the recent years, however, numerous isothermal amplification methods were developed to avoid the need for thermal cycling. The most frequently applied approach seems to be loop-mediated isothermal amplification (LAMP). The great advantage of LAMP is its enormous rate of amplification paired with a very high specificity and low artefact susceptibility. This study presents a straightforward procedure for Plum pox virus (PPV) detection. A modified one-step reverse transcription loop-mediated isothermal amplification protocol of Varga and James is applied to virus suspensions from plant extracts obtained by a simplified and standardised procedure. Gel electrophoresis is substituted by a homogenous colour test upon nucleic acid amplification. This procedure takes only 2.5 h from sampling to result and requires minimal technical equipment. With amplification and visualisation homogenously taking place in non-opened tubes the risk of cross-contamination of subsequent samples by former amplification products via facilities and equipment is strongly minimised. Hence, the Blue LAMP provides a fast and reliable detection of PPV both for single samples and for large-scale surveys.