Detection of pine wood nematodes by droplet digital PCR, qPCR, and RPA-LFA

Abstract The nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease and is one of the most destructive plant-parasitic nematodes worldwide. As there are no effective control measures, early and accurate detection is crucial to prevent disease spread. Here, we developed a droplet digital PCR (ddPCR) assay and a recombinase polymerase amplification-lateral flow assay (RPA-LFA) for detecting B. xylophilus and compared these methods with real-time quantitative PCR (qPCR). The results showed that this ddPCR assay was highly specific for B. xylophilus and could be quantified through the 5S gene copy numbers. Compared to qPCR, ddPCR is more sensitive and achieves absolute quantification. Additionally, the results of the RPA-LFA were 100% consistent with the positive qPCR results but significantly reduced the required reaction time to within 30 min. Together, establishing and combining these two assays can offer an effective method for the early detection of B. xylophilus , i.e., the ddPCR assay represents a promising alternative method for the precise quantitative detection of B. xylophilus, while the RPA-LFA is a simple, rapid and visual method that can be used for rapid detection of B. xylophilus in the field and in resource-limited conditions.