Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification approach that has been used to detect a variety of animal and plant pathogens. However, the RPA assay is rarely used for the molecular diagnosis of plant parasitic nematodes. In this study, we developed RPA assays for the detection of four Meloidogyne spp.; Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The RPA amplification step could be completed at 38 °C in 20 min without a thermal cycling instrument. The RPA assays were able to distinguish these four Meloidogyne spp. from closely related Meloidogyne species and other plant parasitic nematodes. The detection limits of the RPA assays were 10−2, 10−2, 10−1, and 10−1 dilutions of DNA from a single J2 nematode of M. incognita, M. javanica, M. arenaria, and M. enterolobii, which were less sensitive than polymerase chain reaction (PCR) detection methods. In addition, the RPA assays could detect these four Meloidogyne spp. directly from infested tomato roots. The simplicity, rapidity and practicability all indicated that the RPA assay will be an effective tool for molecular diagnosis of plant parasitic nematodes.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Adam, M. A. M., Phillips, M. S., & Blok, V. C. (2007). Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.). Plant Pathology, 56, 190–197.
Berry, S. D., Fargette, M., Spaull, V. W., Morand, S., & Cadet, P. (2008). Detection and quantification of root-knot nematode (Meloidogyne javanica), lesion nematode (Pratylenchus zeae) and dagger nematode (Xiphinema elongatum) parasites of sugarcane using real-time PCR. Molecular and Cellular Probes, 22, 168–176.
Craw, P., & Balachandran, W. (2012). Isothermal nucleic acid amplification technologies for point-of-care diagnostics: A critical review. Lab on a Chip, 12, 2469–2486.
Elling, A. A. (2013). Major emerging problems with minor Meloidogyne species. Phytopathology, 103, 1092–1102.
Euler, M., Wang, Y., Otto, P., Tomaso, H., Escudero, R., Anda, P., Hufert, F. T., & Weidmann, M. (2012). Recombinase polymerase amplification assay for rapid detection of Francisella tularensis. Journal of Clinical Miceobiology, 50, 2234–2238.
Euler, M., Wang, Y., Heidenreich, D., Patel, P., Strohmeier, O., Hakenberg, S., Niedrig, M., Hufert, F. T., & Weidmann, M. (2013). Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents. Journal of Clinical Microbiology, 51, 1110–1117.
Gheysen, G., & Mitchum, M. G. (2011). How nematodes manipulate plant development pathways for infection. Current Opinion in Plant Biology, 14, 415–421.
Ghosh, D. K., Kokane, S. B., Kokane, A. D., Warghane, A. J., Motghare, M. R., Bhose, S., Sharma, A. K., & Reddy, M. K. (2018). Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus". PLoS One, 13, e0208530.
Htay, C., Peng, H., Huang, W., Kong, L., He, W., Holgado, R., & Peng, D. (2016). The development and molecular characterization of a rapid detection method for rice root-knot nematode (Meloidogyne graminicola). European Journal of Plant Pathology, 146, 281–291.
Hu, M., Zhuo, K., & Liao, J. (2011). Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls. Phytopathology, 101, 1270–1277.
Jones, J. T., Haegeman, A., Danchin, E. G., Gaur, H. S., Helder, J., & Jones, M. G. (2013). Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology, 14, 946–961.
Kersting, S., Rausch, V., Bier, F. F., & von Nickisch-Rosenegk, M. (2014). Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malaria Journal, 13, 99.
Kim, J., & Easley, C. J. (2011). Isothermal DNA amplification in bioanalysis: Strategies and applications. Bioanalysis, 3, 227–239.
Lau, H. Y., & Botella, J. R. (2017). Advanced DNA-based point-of-care diagnostic methods for plant diseases detection. Frontiers in Plant Science, 8, 2016.
Lodh, N., Naples, J. M., Bosompem, K. M., Quartey, J., & Shiff, C. J. (2014). Detection of parasite-specific DNA in urine sediment obtained by filtration differentiates between single and mixed infections of Schistosoma mansoni and S. haematobium from endemic areas in Ghana. PLoS One, 9, e91144.
Londono, M. A., Harmon, C. L., & Polston, J. E. (2016). Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics. Virology Journal, 13, 48.
Long, H., Liu, H., & Xu, J. (2006). Development of a PCR diagnostic for the root-knot nematode Meloidogyne enterobbii. Acta Phytopathologica Sinica, 36, 109–115.
Meng, Q., Long, H., & Xu, J. (2004). PCR assays for rapid and sensitive identification of three major root-knot nematodes, Meloidogyne incognita, M. javanica and M. arenaria. Acta Phytopathologica Sinica, 34, 204–210.
Moens, M., Perry, R. N., & Starr, J. L. (2009). Meloidogyne species-a diverse group of novel and important plant parasites. In R. N. Perry, M. Moens, & J. L. Starr (Eds.), Root-knot nematodes (pp. 1–17). Wallingford: CAB International.
Mondal, D., Ghosh, P., Khan, M. A. A., Hossain, F., Bohlken-Fascher, S., Matlashewski, G., et al. (2016). Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay. Parasites & Vectors, 9, 281.
Moore, M. D., & Jaykus, L. A. (2017). Recombinase polymerase amplification: A promising point-of-care detection method for enteric viruses. Future Virology, 12, 421–429.
Nicol, J. M., Turner, S. J., Coyne, D. L., den Nijs, L., Hockland, S., & Tahna Maafi, Z. (2011). Current nematode threats to world agriculture. In J. T. Jones, G. Gheysen, & C. Fenoll (Eds.), Genomics and molecular genetics of plant-nematode interactions (pp. 21–43). Heidelberg: Springer.
Niu, J., Guo, Q., Jian, H., Chen, C., Yang, D., Liu, Q., & Guo, Y. D. (2011). Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots. Crop Protection, 30, 1063–1069.
Niu, J., Jian, H., Guo, Q., Chen, C., Wang, X., Liu, Q., et al. (2012). Evaluation of loop-mediated isothermal amplification (LAMP) assay based on 5S rDNA-IGS2 regions for detecting Meloidogyne enterolobii. Plant Pathology, 61, 809–819.
Peng, H., Long, H., Huang, W., Liu, J., Cui, J., Kong, L., Hu, X., Gu, J., & Peng, D. (2017). Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology. Scientific Reports, 7, 44853.
Piepenburg, O., Williams, C. H., Stemple, D. L., & Armes, N. A. (2006). DNA detection using recombination proteins. PLoS Biology, 4, 1115–1121.
Powers, T. O., & Harris, T. S. (1993). A polymerase chain reaction method for identification of five major Meloidogyne species. Journal of Nematology, 25, 1–6.
Rahman, S. A. S. A., Mohamed, Z., Othman, R. Y., Swennen, R., Panis, B., De Waele, D., et al. (2010). In planta PCR-based detection of early infection of plant-parasitic nematodes in the roots: A step towards the understanding of infection and plant defense. European Journal of Plant Pathology, 128, 343–351.
Randig, O., Bongiovanni, M., Carneiro, R. M., & Castagnone-Serno, P. (2002). Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species. Genome, 45, 862–870.
Reid, M. S., Le, X. C., & Zhang, H. (2018). Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities. Angewandte Chemie International Edition, 57, 11856–11866. https://doi.org/10.1002/anie.201712217.
Subbotin, S. A. (2018). Recombinase polymerase amplification assay for rapid detection of the root-knot nematode Meloidogyne enterolobii. Nematology, 21, 243–251.
Tigano, M., Siqueira, K. D., Castagnone-Sereno, P., Mulet, K., Queiroz, P., Santos, M. D., et al. (2010). Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava-damaging species. Plant Pathology, 59, 1054–1061.
Toyota, K., Shirakashi, T., Sato, E., Wada, S., & Min, Y. Y. (2008). Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita. Soil Science and Plant Nutrition, 54, 72–76.
Zaghloul, H., & El-Shahat, M. (2014). Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis. World Journal of Hepatology, 6, 916–922.
Zijlstra, C., Donkers-Venne, D. T. H. M., & Fargette, M. (2000). Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence characterized amplified region (SCAR) based PCR assays. Nematology, 2, 847–853.
Zou, Y., Mason, M. G., Wang, Y., Wee, E., Turin, C., Blackall, P. J., et al. (2018). Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biology, 16, e2003916.
This work was supported by the National Natural Science Foundation of China (Grant No. 18234004), the Natural Science Foundation of Anhui Province (Grant No. 1808085QC80), and the Natural Science Foundation of Anhui Provincial Department of Education (Grant No. KJ2018A0147).
Conflict of interest
The authors declare no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the author.
Informed consent was not applicable to this article since no information regarding individual participants was included in the study.
About this article
Cite this article
Ju, Y., Lin, Y., Yang, G. et al. Development of recombinase polymerase amplification assay for rapid detection of Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. Eur J Plant Pathol 155, 1155–1163 (2019). https://doi.org/10.1007/s10658-019-01844-6
- Recombinase polymerase amplification
- Molecular diagnosis