Abstract
Detection of root-knot nematodes (RKN) in soil and plant samples is crucial to prevent its spread and select effective control measures. In this study, Recombinase Polymerase Amplification (RPA) assays using lateral flow dipsticks (LF-RPA) and real-time fluorescence detection (real-time RPA) were developed to detect the RKN species from tropical complex using a group-specific primer-probe set and Meloidogyne javanica using a species-specific primer-probe set. The results of the real time RPA assays in series of crude nematode extracts showed reliable detection within 16 min with a sensitivity of 1/100 of a second-stage juvenile in a reaction tube. The results of the LF-RPA assays showed reliable detection within 30 min with a sensitivity of 1/20 to 1/100 of a second-stage juvenile and 1/10 of a female in a reaction tube. Real-time RPA and LF-RPA assays are highly specific and can identify their target DNA in mixtures with other nematodes and plant tissues. LF-RPA assay has great potential for diagnosing RKN in the lab, field or in areas with a minimal laboratory infrastructure.
Highlights
Detection of root-knot nematodes (RKN) in soil and plant samples is crucial to prevent its spread and select effective control measures
Recombinase Polymerase Amplification (RPA) assays with a gel visualization of amplicons for species diagnostics of M. javanica, M. arenaria, M. incognita and M. enterolobii have been developed by Ju et al (2019)
The goal of this study was to develop real-time RPA and LF-RPA assays with group-specific primersprobe sets for the detection of the major RKN be longing to the tropical RKN complex (M. arenaria, M. floridensis, M. hispanica, M. incognita, M. javanica and M. paranaensis) and species-specific primersprobe set the detection of M. javanica only
Summary
Detection of root-knot nematodes (RKN) in soil and plant samples is crucial to prevent its spread and select effective control measures. Recombinase Polymerase Amplification (RPA) assays using lateral flow dipsticks (LF-RPA) and real-time fluorescence detection (real-time RPA) were developed to detect the RKN species from tropical complex using a group-specific primer-probe set and Meloidogyne javanica using a species-specific primer-probe set. The results of the real time RPA assays in series of crude nematode extracts showed reliable detection within 16 min with a sensitivity of 1/100 of a second-stage juvenile in a reaction tube. RPA has several advantages over PCRbased methods of plant-parasitic nematode detection: (i) does not require thermal cycling and can be used in areas with minimal laboratory infrastructure and run by personals with minimal technical experience; (ii) in 10 or more times sensitive than PCR; (iii) does not require DNA extraction in sample processing; (iv) amplicons may be detected at endpoint or in real-time during 8 to 30 min. RPA assays for detection of the northern RKN, M. hapla were developed by Song et al (2021) and Subbotin and Burbridge (2021)
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