Rapid detection of Pratylenchus coffeae using recombinase polymerase amplification assay with lateral flow dipsticks

Abstract

Pratylenchus coffeae is a migratory endoparasitic nematode that causes root rot in a wide range of crops, resulting in enormous financial losses each year. The rapid and accurate detection of P. coffeae is crucial for controlling and combating the disease of root rot. We designed species-specific primers and probes based on the parasite's rDNA-ITS sequence and developed a rapid P. coffeae detection method employing a recombinase polymerase amplification (RPA) assay. The RPA reaction can be completed in 5–30 min at 38 °C without the application of a thermal cycling instrument and has an optimal time of 25 min. The amplification results can be directly observed on the test strip in 3–5 min. We validated the RPA assay and showed that it accurately detects P. coffeae in infested host tissues and soil samples, including nematodes from complex and diverse geographic populations. The RPA-lateral flow dipstick(RPA-LFD)assay has a detection limit of 0.01 ng/μL pure gDNA, making it 100 times more sensitive than traditional polymerase chain reaction assays. The results indicate that the RPA assay is a sensitive, rapid, practical, and visual method for the rapid detection and diagnosis of P. coffeae from infested fields and the RPA-LFD assay is accessible for point-of-service or lab-in-a-suitcase diagnosis.