Development of a Recombinase Polymerase Amplification Method Combined with a Lateral Flow Dipstick Assay for Rapid Detection of the Larch Pathogen Neofusicoccum laricinum.

Abstract

Neofusicoccum laricinum, an important pathogenic species, causes shoot blight of larch. In China, large areas of Larix principis-rupprechtii forests are threatened by this pathogen. Currently, this pathogen is on the list of quarantine pests in China. Because of the widespread and severe damage caused by N. laricinum, a reliable and accurate diagnostic tool is urgently needed. In this study, we first identified Nlar12009 as a N. laricinum-specific gene through genomic sequence data and bioinformatic analysis. Specific primer pairs and DNA probes were designed to detect the target pathogen using a novel recombinase polymerase amplification assay with a lateral flow dipstick (RPA-LFD) method. We optimized the RPA-LFD assay to ensure high specificity to N. laricinum. Our results showed that the assay exclusively detected N. laricinum isolates with no cross-reaction with other isolates of fungal and oomycete species and nematodes. Furthermore, our detection technique exhibited a 10-fold higher sensitivity (10 fg/ml) than conventional polymerase chain reaction for N. laricinum detection. Our developed RPA-LFD assay is proved to be a highly specific, sensitive, time-saving, and convenient method for the diagnosis of N. laricinum and shows great potential in field application.