Skip to main content

Recombinase Polymerase Amplification (RPA) for the Rapid Isothermal Detection of Spongospora subterranea f. sp. subterranea and Potato Mop-Top Virus

American Journal of Potato Research volume 96, pages 617–624 (2019)Cite this article

Abstract

The amplification of specific nucleic acid sequences with high specificity and sensitivity is an essential technique for pathogen detection. Recombinase polymerase amplification (RPA) is a rapid isothermal amplification method. Here, we demonstrate the end-point and real-time detection of Spongospora subterranea f. sp. subterranea (Sss) using RPA and potato mop-top virus (PMTV) using reverse transcription (RT)-RPA. Oligonucleotide primers were designed for amplification that target the internal transcribed spacer 1 (ITS1) region and the coat protein readthrough (CP-RT) domain for the detection of Sss and PMTV, respectively. Our data showed that real-time RPA can detect 100 of Sss sporosori per gram of soil, while real-time RT-RPA could detect in ~1 ng total RNA of the PMTV-infected tuber. For Sss detection, the R2 value for real-time RPA and real-time PCR was 98.0% by linear regression analysis in the concentration range of 100–34,000 sporosori per gram of soil. The developed RPA assay here may provide a useful alternative tool for the rapid, simple and reliable detection of Sss and PMTV in diagnostic laboratories and in-field testing.

Resumen

La amplificación de secuencias específicas de ácido nucléico con alta especificidad y sensibilidad, es una técnica esencial para la detección de patógenos. La ampliación de polimerasa de recombinación (RPA) es un método rápido de amplificación isotérmica. Aquí demostramos la detección final, y de tiempo real, de Spongospora subterranea f. sp. subterranea (Sss), usando RPA, y del virus del trapeador apical de la papa (PMTV), usando transcripción reversa (RT)-RPA. Los oligonucleótidos iniciadores se designaron para la amplificación que diera con el objetivo de la región del espaciador interno transcrito 1 (ITS1) y del dominio de la lectura completa de la cubierta proteica (CP-RT) para la detección de Sss y PMTV, respectivamente. Nuestros datos mostraron que la RPA de tiempo real puede detectar 100 esporosori de Sss por gramo de suelo, mientras que RT-RPA de tiempo real pudo detectar ~1 ng del ARN total en el tubérculo infectado con PMTV. Para la detección de Sss, el valor de R2 para RPA de tiempo real y PCR de tiempo real fue de 98.0% mediante el análisis de regresión lineal en la amplitud de concentración de 100–34,000 esporosori por gramo de suelo. El ensayo de RPA desarrollado pudiera proporcionar una herramienta alternativa útil para la detección simple y confiable de Sss y PMTV en laboratorios de diagnóstico y en pruebas de campo.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Bentahir, M., J. Ambroise, C. Delcorps, P. Pilo, and J.-L. Gala. 2018. Sensitive and specific Recombinase polymerase amplification assays for fast screening, detection, and identification of bacillus anthracis in a field setting. Applied and Environmental Microbiology 84: e00506–e00518.

    Article  CAS  Google Scholar 

  • Budziszewska, M., P. Wieczorek, K. Nowaczyk, N. Borodynko, H. Pospieszny, and A. Obrêpalska-Stêplowska. 2010. First report of potato mop-top virus on potato in Poland. Plant Disease 94: 920–920.

    Article  CAS  Google Scholar 

  • Bulman, S., J.M. Candy, M. Fiers, R. Lister, A.J. Conner, and C.C. Eady. 2011. Genomics of biotrophic, plant-infecting Plasmodiophorids using in vitro dual cultures. Protist 162: 449–461.

    Article  CAS  Google Scholar 

  • Calvert, E.L. 1968. The reaction of potato varieties to potato mop-top virus. Record of Agricultural Research. Ministry of Agriculture Northern Ireland 17: 31–40.

    Google Scholar 

  • Ciaghi, S., S. Neuhauser, and A. Schwelm. 2018. Draft genome resource for the potato powdery scab pathogen Spongospora subterranea. Molecular Plant-Microbe Interactions 31: 1227–1229.

    Article  CAS  Google Scholar 

  • Crosslin, J.M. 2011. First report of potato mop-top virus on potatoes in Washington state. Plant Disease 95: 1483–1483.

    Article  CAS  Google Scholar 

  • DeShields, J.B., R.A. Bomberger, J.W. Woodhall, D.L. Wheeler, N. Moroz, D.A. Johnson, and K. Tanaka. 2018. On-site molecular detection of soil-borne phytopathogens using a portable real-time PCR system. Journal of Visualized Experiments: e56891.

  • Down, G.J., L.J. Grenville, and J.M. Clarkson. 2002. Phylogenetic analysis of Spongospora and implications for the taxonomic status of the plasmodiophorids. Mycological Research 106: 1060–1065.

    Article  CAS  Google Scholar 

  • Doyle, J.J. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13–15.

    Google Scholar 

  • Euler, M., Y. Wang, P. Otto, H. Tomaso, R. Escudero, P. Anda, F.T. Hufert, and M. Weidmann. 2012. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis. Journal of Clinical Microbiology 50: 2234–2238.

    Article  CAS  Google Scholar 

  • Gau, R.D., U. Merz, R.E. Falloon, and P.C. Brunner. 2013. Global genetics and invasion history of the potato powdery scab pathogen, Spongospora subterranea f.sp. subterranea. PLoS One 8: e67944.

    Article  CAS  Google Scholar 

  • Gilchrist, E., J. Soler, U. Merz, and S. Reynaldi. 2011. Powdery scab effect on the potato Solanum tuberosum ssp. andigena growth and yield. Tropical Plant Pathology 36: 350–355.

    Article  Google Scholar 

  • Gill, P., and A. Ghaemi. 2008. Nucleic acid isothermal amplification technologies: A review. Nucleosides, Nucleotides & Nucleic Acids 27: 224–243.

    Article  CAS  Google Scholar 

  • Gutiérrez Sánchez, P.A., J.F. Alzate, and M. Marín Montoya. 2014. Analysis of carbohydrate metabolism genes of Spongospora subterranea using 454 pyrosequencing. Revista Facultad Nacional de Agronomía Medellín 67: 7247–7260.

    Article  Google Scholar 

  • Hu, X., V. Dickison, Y. Lei, C. He, M. Singh, Y. Yang, X. Xiong, and X. Nie. 2016. Molecular characterization of potato mop-top virus isolates from China and Canada and development of RT-PCR differentiation of two sequence variant groups. Canadian Journal of Plant Pathology 38: 231–242.

    Article  CAS  Google Scholar 

  • Jones, R.A.C., and B.D. Harrison. 1969. The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh. The Annals of Applied Biology 63: 1–17.

    Article  Google Scholar 

  • Kumar, G.N.M., S. Iyer, and N.R. Knowles. 2007. Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues. Journal of Agricultural and Food Chemistry 55: 1674–1678.

    Article  CAS  Google Scholar 

  • Mekuria, T.A., S. Zhang, and K.C. Eastwell. 2014. Rapid and sensitive detection of little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification. Journal of Virological Methods 205: 24–30.

    Article  CAS  Google Scholar 

  • Moroz, N., K.R. Fritch, M.J. Marcec, D. Tripathi, A. Smertenko, and K. Tanaka. 2017. Extracellular Alkalinization as a defense response in potato cells. Frontiers in Plant Science 8: 32.

    Article  Google Scholar 

  • Morse, W.J. 1912. Does the potato scab organism survive passage through the digestive tract of domestic animals? Phytopathology 2: 146–149.

    Google Scholar 

  • Piepenburg, O., C.H. Williams, D.L. Stemple, and N.A. Armes. 2006. DNA detection using recombination proteins. PLoS Biology 4: e204.

    Article  Google Scholar 

  • Qian, W., Y. Lu, Y. Meng, Z. Ye, L. Wang, R. Wang, Q. Zheng, H. Wu, and J. Wu. 2018. Field detection of Citrus Huanglongbing associated with ‘Candidatus Liberibacter Asiaticus’ by recombinase polymerase amplification within 15 min. Journal of Agricultural and Food Chemistry 66: 5473–5480.

    Article  CAS  Google Scholar 

  • Rohrman, B., and R. Richards-Kortum. 2015. Inhibition of recombinase polymerase amplification by background DNA: A lateral flow-based method for enriching target DNA. Analytical Chemistry 87: 1963–1967.

    Article  CAS  Google Scholar 

  • Rojas, J.A., T.D. Miles, M.D. Coffey, F.N. Martin, and M.I. Chilvers. 2017. Development and application of qPCR and RPA genus- and species-specific detection of Phytophthora sojae and P. sansomeana root rot pathogens of soybean. Plant Disease 101: 1171–1181.

    Article  CAS  Google Scholar 

  • Silva, G., M. Bömer, C. Nkere, P.L. Kumar, and S.E. Seal. 2015. Rapid and specific detection of yam mosaic virus by reverse-transcription recombinase polymerase amplification. Journal of Virological Methods 222: 138–144.

    Article  CAS  Google Scholar 

  • Silva, G., J. Oyekanmi, C. Nkere, M. Bömer, P.L. Kumar, and S.E. Seal. 2018. Rapid detection of potyviruses from crude plant extracts. Analytical Biochemistry 546: 17–22.

    Article  CAS  Google Scholar 

  • Strayer-Scherer, A., J.B. Jones, and M.L. Paret. 2018. Recombinase polymerase amplification assay for field detection of tomato bacterial spot pathogens. Phytopathology 109: 690–700.

    Article  Google Scholar 

  • Tenorio, J., Y. Franco, C. Chuquillanqui, R.A. Owens, and L.F. Salazar. 2006. Reaction of potato varieties to potato mop-top virus infection in the Andes. American Journal of Potato Research 83: 423–431.

    Article  CAS  Google Scholar 

  • Valkonen, J.P.T. 2015. Elucidation of virus-host interactions to enhance resistance breeding for control of virus diseases in potato. Breeding Science 65: 69–76.

    Article  CAS  Google Scholar 

  • Xu, H., T.-L. DeHaan, and S.H. De Boer. 2004. Detection and confirmation of potato mop-top virus in potatoes produced in the United States and Canada. Plant Disease 88: 363–367.

    Article  CAS  Google Scholar 

  • Zhang, S.L., M. Ravelonandro, P. Russell, N. McOwen, P. Briard, S. Bohannon, and A. Vrient. 2014. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(R) using reverse transcription-recombinase polymerase amplification. Journal of Virological Methods 207: 114–120.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Drs. Richard Quick and Chuck Brown (USDA-ARS) for providing PMTV-infected tubers. Special thanks to Jacquie van der Waals (University of Pretoria) for finding us key literature on a topic. This research was supported by the Northwest Potato Research Consortium, the Washington State Department of Agriculture - Specialty Crop Block Grant Program (grant no. K1764), and USDA National Institute of Food and Agriculture (AFRI grant award no. 2019-67013-29963 and Hatch project no. 1015621) to K.T., and also by the Mexican National Council of Science and Technology (CONACyT) scholarship program to G.A.M.R. PPNS No. 0773, Department of Plant Pathology, College of Agriculture, Human and Natural Resource Sciences, Agricultural Research Center, Washington State University, Pullman, WA, 99164–6430, USA.

Author information

Author notes

  1. Joseph B. DeShields and Natalia Moroz contributed equally to this work.

Authors and Affiliations

  1. Department of Plant Pathology, Washington State University, Pullman, WA, USA

    Joseph B. DeShields, Natalia Moroz, Lauren E. Braley & Kiwamu Tanaka

  2. Unidad de Investigación en Ambiente y Salud, Universidad Autónoma de Occidente, Los Mochis, Sinaloa, Mexico

    Guadalupe Arlene Mora-Romero

Authors
  1. Joseph B. DeShields
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natalia Moroz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lauren E. Braley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guadalupe Arlene Mora-Romero
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kiwamu Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kiwamu Tanaka.

Electronic Supplementary Material

ESM 1

(PDF 1.25 MB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

DeShields, J.B., Moroz, N., Braley, L.E. et al. Recombinase Polymerase Amplification (RPA) for the Rapid Isothermal Detection of Spongospora subterranea f. sp. subterranea and Potato Mop-Top Virus. Am. J. Potato Res. 96, 617–624 (2019). https://doi.org/10.1007/s12230-019-09750-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12230-019-09750-7

Keywords

  • Isothermal amplification-based pathogen detection
  • Recombinase polymerase amplification (RPA)
  • Real-time RPA
  • Spongospora subterranea f. sp. subterranea (Sss)
  • Potato mop-top virus (PMTV)