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A Multiplex Reverse Transcription (RT) High-Fidelity PCR Protocol for the Detection of Six Viruses that Cause Potato Tuber Necrosis

American Journal of Potato Research Aims and scope Submit manuscript

Abstract

Viruses that cause necrotic symptoms in potato tubers can be difficult to distinguish based on symptoms and frequently require multiple molecular tests to identify the pathogen. In this study, a multiplex RT PCR high fidelity PCR protocol was developed using previously validated primers that could accurately detect six important potato viruses and detect multiple viruses in a single sample simultaneously. To test the protocol, 53 tubers previously tested using conventional PCR were retested using this multiplex protocol and blindly evaluated. In every case, the multiplex PCR results were consistent with previous findings. In addition, the multiplex PCR protocol also detected several mixed infections that were previously undetected. The results demonstrate that this technique could be a valuable assay not only for diagnostic laboratories, but also for seed certification and regulatory agency laboratories to diagnose the virus(es) that may be present in tubers with surface or internal necrotic symptoms.

Resumen

Los virus que causan síntomas necróticos en tubérculos de papa pueden ser difíciles de distinguir con base en los síntomas, y frecuentemente se requiere de múltiples pruebas moleculares para identificar al patógeno. En este estudio, se desarrolló un protocolo múltiple de RT PCR de alta fidelidad de PCR utilizando iniciadores previamente validados que pudieran detectar con precisión seis virus importantes de papa y múltiples virus en una única muestra simultáneamente. Para probar el protocolo, se analizaron nuevamente 53 tubérculos que habían sido probados previamente usando la PCR convencional, utilizando este protocolo múltiple con evaluación ciega. En cada caso, los resultados de PCR múltiple fueron consistentes con lo que se encontró previamente. Además, el protocolo de PCR múltiple también detectó algunas mezclas de infecciones que previamente no se detectaron. Los resultados demuestran que esta técnica pudiera ser un ensayo valioso no solo para laboratorios de diagnóstico, sino también para certificación de semilla y para laboratorios de agencias de regulación para diagnosticar el (los) virus que pudieran estar presentes en tubérculos con síntomas necróticos superficiales o internos.

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Authors and Affiliations

  1. Department of Botany & Plant Pathology, Hermiston Agricultural Research and Extension Center, Oregon State University, Hermiston, OR, USA

    Robert A. Cating, Cassandra N. Funke, Navneet Kaur, Philip B. Hamm & Ken E. Frost

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  1. Robert A. Cating
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  2. Cassandra N. Funke
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  3. Navneet Kaur
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  4. Philip B. Hamm
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  5. Ken E. Frost
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Correspondence to Robert A. Cating.

Additional information

Cating, R. A., C.N. Funke, N. Kaur, P.B. Hamm, and K.E. Frost, 2015. A multiplex reverse transcription (RT) high-fidelity PCR protocol for the simultaneous detection of six viruses that cause potato tuber necrosis. American Journal of Potato Research 92:______.

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Cating, R.A., Funke, C.N., Kaur, N. et al. A Multiplex Reverse Transcription (RT) High-Fidelity PCR Protocol for the Detection of Six Viruses that Cause Potato Tuber Necrosis. Am. J. Potato Res. 92, 536–540 (2015). https://doi.org/10.1007/s12230-015-9457-5

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  • DOI: https://doi.org/10.1007/s12230-015-9457-5

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