Recombinase-Aided Amplification Combined with Lateral Flow Dipstick for the Rapid Detection of Pyrrhoderma noxium

Pyrrhoderma noxium is an entry quarantine tree pathogenic fungus in China, which causes severe root rot and the death of many tree species, resulting in huge landscape, ecological, and economic losses. P. noxium is a soilborne plant pathogen, which makes it difficult to observe the symptoms of infected trees in the early stage. In this study, a rapid, specific, and visible detection method for P. noxium was established by integrating recombinase-aided amplification (RAA) with a lateral flow dipstick (LFD), called RAA-LFD. First, we screened out a specific gene of P. noxium, PnEf1, as the target for RAA amplification by using bioinformatic analysis. Then, a pair of specific RAA primers and a probe with high efficiency were designed. Finally, the RAA amplification results were visualized with the LFD. The RAA-LFD detection system of P. noxium was performed at 37°C (body temperature) and did not rely on precise instruments. Remarkably, the detection limit of this system was 100 fg/μl and 5.5 copies with P. noxium gDNA as the template, which was significantly superior to the reported methods. Importantly, the examination results could be visualized in about 20 min with the naked eye, which indicated a promising application in the field detection of P. noxium. In conclusion, the established RAA-LFD detection system of P. noxium was rapid, highly sensitive and specific, easily observed, and simply operated, which provides strong technical support for the early warning and detection of P. noxium in the field.