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Updated: Dec 30, 2025
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Evaluation of Recombinase Polymerase Amplification Assay for Detecting Meloidogyne javanica
Yuan-Kai Chi1, Wei Zhao1, Meng-di Ye2
1Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei, China.
Abstract:
Meloidogyne javanica is one of the most widespread and economically important nematodes in many countries, including China. In this study, a recombinase polymerase amplification (RPA) assay was evaluated for the detection of M. javanica based on the sequences of a sequence-characterized amplified regions marker gene segment. The RPA assay specifically detected M. javanica from individual juvenile or adult female, M. javanica-induced galls, and nematodes in the soil samples. The detection limit of M. javanica RPA assay was 1 pg of purified genomic DNA, 0.01 adult female, or 0.1 second-stage juvenile, which was 10 times more sensitive than conventional PCR assay. Furthermore, combined with lateral flow dipstick (LFD), a visual detection method of LFD-RPA assay was developed, which is suitable for onsite surveys and routine diagnostics. Results indicate that the RPA assay is rapid, sensitive, and reliable for detection and molecular identification of M. javanica.
Insights
A new recombinase polymerase amplification (RPA) assay rapidly and reliably detects Meloidogyne javanica, a damaging plant-parasitic nematode. This sensitive method, suitable for onsite surveys, offers improved detection over conventional PCR.
Area of Science:
- Plant Pathology
- Molecular Diagnostics
- Nematology
Background:
- Meloidogyne javanica is a globally significant plant-parasitic nematode causing substantial economic losses.
- Accurate and rapid detection methods are crucial for managing M. javanica infestations in agriculture.
- Existing diagnostic techniques can be time-consuming or lack sufficient sensitivity for early detection.
Purpose of the Study:
- To evaluate a recombinase polymerase amplification (RPA) assay for the specific detection of Meloidogyne javanica.
- To assess the sensitivity and reliability of the RPA assay compared to conventional PCR.
- To develop a visual detection method for onsite application using lateral flow dipsticks (LFDs).
Main Methods:
- Developed and optimized an RPA assay targeting a sequence-characterized amplified regions marker gene segment of M. javanica.
- Tested the specificity of the RPA assay using various life stages (juveniles, adult females) and sample types (galls, soil).
- Determined the detection limit of the RPA assay and compared it with conventional PCR.
- Integrated the RPA assay with LFDs to create a visual, onsite detection system (LFD-RPA).
Main Results:
- The RPA assay specifically detected M. javanica from individual nematodes, galls, and soil samples.
- The detection limit was as low as 1 pg genomic DNA, 0.01 adult female, or 0.1 second-stage juvenile.
- The RPA assay demonstrated 10-fold higher sensitivity than conventional PCR.
- The LFD-RPA assay provided a rapid, visual, and reliable method for onsite detection.
Conclusions:
- The developed RPA assay is a rapid, highly sensitive, and reliable tool for the molecular detection and identification of Meloidogyne javanica.
- The LFD-RPA assay offers a practical solution for field-based diagnostics and routine monitoring of M. javanica.
- This molecular approach can significantly aid in the timely management of M. javanica to mitigate crop losses.
