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

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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.

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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.

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  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

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  1. Joseph B. DeShields
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  2. Natalia Moroz
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  3. Lauren E. Braley
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  4. Guadalupe Arlene Mora-Romero
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  5. Kiwamu Tanaka
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Correspondence to Kiwamu Tanaka.

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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

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