Loop-Mediated Isothermal Amplification (LAMP)-Based Turn on Fluorescent Paper (ToFP) Device for Detecting Rosellinia necatrix

Abstract

White root rot (WRR) disease caused by Rosellinia necatrix, a fungal pathogen, results in severe damage to various fruit trees, decreasing their marketability. Regular monitoring is a major process because the pathogen can remain in the soil around the host for a long time. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and efficient amplification technology of nucleic acids (DNA or RNA) that can be performed at constant temperatures. Thus, it has been spotlighted as a useful tool for detecting several infectious agents. In the present study, LAMP-based Turn-on Fluorescent Paper (ToFP) devices were designed and applied to detect R. necatrix. LAMP conditions were optimized and found to be optimal at a reaction temperature (62 °C) and a reaction time (30 minutes). These reaction conditions were confirmed by applying them to infectious soil samples collected from the field. The limitation of detection was identified as 10 fg of genomic DNA under optimized LAMP conditions. These LAMP-based ToFP devices were generated with easily available stationery materials and the utility of these devices to analyze the LAMP results were confirmed through several experiments on a total of 14 field samples. The results showed that the developed LAMP-based detection system was very sensitive and had the advantages of rapid detection and high availability in the field.