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Rapid Colorimetric Detection of the Fungal Phytopathogen Synchytrium endobioticum Using Cyanine dye-Indicated PNA Hybridization

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Abstract

Synchytrium endobioticum is a parasitic fungus that is considered to be the most important potato pathogen worldwide. Spread of the fungus can result in catastrophic impacts to agriculture and economy, as it is very infective and can survive in spore form for over 70 years. To date, the gold standard for S. endobioticum identification relies on visual classification by highly trained personnel. This paper presents the colorimetric detection of S. endobioticum from potato wart samples using a peptide nucleic acid (PNA) probe and a cyanine dye. A segment of the 18S ribosomal RNA gene was sequenced for several S. endobioticum pathotypes and used to design a species-specific PNA probe. The probe was used in a rapid colorimetric hybridization assay that detected RNA from infected wart tissue down to 10−17 mol within 15 min. This technique demonstrates potential for rapid identification of potato wart in non-laboratory settings with minimally trained staff.

Resumen

Synchytrium endobioticum es un hongo parasítico considerado como el patógeno de la papa más importante en el mundo. La dispersión del hongo pudiera resultar en impactos catastróficos a la agricultura y la economía, porque es muy infectivo y puede sobrevivir como espora por más de setenta años. A la fecha, el estándar dorado para la identificación de S. endobioticum se respalda en clasificación visual por personal altamente entrenado. Este artículo presenta la detección colorimétrica de S. endobioticum de muestras de papa con verruga negra usando una sonda de péptido de ácido nucléico (PNA) y un colorante de cianina. Se secuenció un segmento de RNA ribosomal de 18 s para varios patotipos de S. endobioticum y se usaron para diseñar una sonda de PNA específica para la especie. Se usó la sonda en un ensayo rápido de hibridación colorimétrica que detectó el RNA de tejido infectado con verruga negra hasta tan bajo como 10–17 mol dentro de 15 minutos. Esta técnica demuestra potencial para identificación rápida de la verruga negra en condiciones ajenas al laboratorio con personal mínimamente entrenado.

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Authors and Affiliations

  1. Graduate School of Biomedical Science and Engineering, ESRB-LASST, University of Maine, Orono, ME, 04469, USA

    Janice Duy, Rosemary L. Smith, Scott D. Collins & Laurie B. Connell

  2. U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St. Fort Detrick, Frederick, MD, 21702, USA

    Janice Duy

  3. Electrical and Computer Engineering Department, Barrows Hall, University of Maine, Orono, ME, 04469, USA

    Rosemary L. Smith

  4. Department of Chemistry, Aubert Hall, University of Maine, Orono, ME, 04469, USA

    Scott D. Collins

  5. School of Marine Sciences, Aubert Hall, University of Maine, Orono, ME, 04469, USA

    Laurie B. Connell

  6. Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME, 04469, USA

    Laurie B. Connell

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  1. Janice Duy
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  2. Rosemary L. Smith
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  4. Laurie B. Connell
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Correspondence to Janice Duy.

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Duy, J., Smith, R.L., Collins, S.D. et al. Rapid Colorimetric Detection of the Fungal Phytopathogen Synchytrium endobioticum Using Cyanine dye-Indicated PNA Hybridization. Am. J. Potato Res. 92, 398–409 (2015). https://doi.org/10.1007/s12230-015-9450-z

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