Development of nucleic acid testing technology for quarantine pest Globodera rostochiensis based on a species-specific DNA fragment

Background Globodera rostochiensis , a potato cyst nematode (PCN) species, is distributed in potato-growing regions across Europe, Asia, Africa, Oceania, and the Americas. Recognized as a quarantine pest under international phytosanitary frameworks, its detection is critical for biosecurity. Methods This study aimed to identify a G. rostochiensis -specific DNA fragment using comparative genomics and develop both a polymerase chain reaction (PCR)-based detection assay and a recombinase-aided amplification (RAA)-based isothermal detection assay. Results A 3,572 bp species-specific DNA fragment was identified. Based on this marker, a conventional PCR diagnostic assay and a recombinase-aided amplification coupled with a lateral flow dipstick (RAA-LFD) diagnostic assay were successfully developed. The PCR primer pair GroF1/GroR1 exhibited a detection limit of 200 copies and a sensitivity of 10 copies/μL (5.6 × 10 −2 fg/μL) when amplified over 32 cycles within a 20 μL PCR reaction volume. Three independent experiments demonstrated that DNA from a single larva could be stably detected using the PCR assay. Following reactions with a 25 μL RAA volume and temperature of 37.5 °C for 10, 15, 20, 25, and 30 min, the detection limit progressively decreased from 5.75 × 10 4 to 5.75 × 10 0 copies. The corresponding sensitivities ranged from 2.3 × 10 3 copies/μL to 2.3 × 10 −1 copies/μL. Across independent experiments, the RAA assay consistently detected DNA equivalent to 10 −2 of a single larva. The RAA-LFD assay achieved 100% diagnostic sensitivity for the detection of G. rostochiensis in infested field soil samples following a 25 min isothermal amplification at 37.5°C. The established RAA-LFD molecular diagnostic assay exhibits marked potential for the field-deployable detection of G. rostochiensis in root zone soil samples, offering rapid turnaround and simplified procedural requirements.