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Simultaneous detection and identification of Pepino mosaic virus (PepMV) isolates by multiplex one-step RT-PCR

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Abstract

A RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1 and Chilean 2/US2 groups, followed by a restriction analysis that allowed the separation of the European, Peruvian, Chilean 2 and US2 isolates (patent pending). The multiplex RT-PCR reaction was performed by a mix of six primers that amplified a part of the RNA-dependent RNA polymerase gene of PepMV plus an internal control. Amplifications resulted in a 980 bp, 703 bp or 549 bp PCR product for European/Peruvian, Chilean 1/US1 or Chilean 2/US2 groups, respectively. For the identification of the isolates present within the European/Peruvian and Chilean 2/US2 groups, the amplified PCR fragments were directly digested with SacI enzyme. The multiplex RT-PCR method presented higher sensitivity to detect CH1/US1 isolates in field samples than the RFLP-PCR method described by Hanssen et al. (European Journal of Plant Pathology 121:131–146, 2008). The detection limit observed with the multiplex RT-PCR was equal to or 3,125 times higher when compared to single RT-PCR or ELISA-DAS and molecular hybridisation methods, respectively. The use of the multiplex RT-PCR method in routine analysis of field tomato samples allowed the detection of 36.2 and 33.4% more positives when compared to the serological and molecular hybridisation methods, respectively, and the identification of plants infected with one, two or three isolates of PepMV.

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Acknowledgements

This work was supported by grants AGL2005-06682-C03-01 and BIO2005-07331-C02-01 from the Ministerio de Educación y Ciencia (MEC, Spain). We thank H. Pospieszny (Institute of Plant Protection, Poznań, Poland) and M. Aranda (CEBAS-CSIC, Murcia, Spain) for providing us with the PK and Sp-13 isolates of PepMV, respectively. We thank A. Espino, R. Martín (Sanidad Vegetal, Canarias) and M. Juarez (Universidad Miguel Hernández, Orihuela) for their assistance in collecting the field samples. We thank Lorena Corachán and Lorena Latorre for excellent technical assistance and Graciela Martínez for reviewing the English text.

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Authors and Affiliations

  1. Instituto Agroforestal Mediterráneo (IAM), Universidad Politécnica de Valencia (UPV), Camino de Vera s/n, 46022, Valencia, Spain

    Ana Alfaro-Fernández, María del Carmen Cebrián, María del Carmen Córdoba-Sellés & Concepción Jordá

  2. Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia UPV-CSIC, Avda. de los Naranjos, s/n, 46022, Valencia, Spain

    Jesús Ángel Sánchez-Navarro & Vicente Pallás

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  1. Ana Alfaro-Fernández
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Correspondence to Ana Alfaro-Fernández.

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Alfaro-Fernández, A., Sánchez-Navarro, J.Á., Cebrián, M.C. et al. Simultaneous detection and identification of Pepino mosaic virus (PepMV) isolates by multiplex one-step RT-PCR. Eur J Plant Pathol 125, 143–158 (2009). https://doi.org/10.1007/s10658-009-9466-7

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