Simultaneous detection and identification of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> (PepMV) isolates by multiplex one-step RT-PCR |
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Authors: | Ana Alfaro-Fernández Jesús Ángel Sánchez-Navarro María del Carmen Cebrián María del Carmen Córdoba-Sellés Vicente Pallás Concepción Jordá |
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Institution: | (1) Instituto Agroforestal Mediterráneo (IAM), Universidad Politécnica de Valencia (UPV), Camino de Vera s/n, 46022 Valencia, Spain;(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 |
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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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Keywords: | ELISA Dot-blot hybridisation Multiplex RT-PCR PepMV genotypes Simultaneous identification |
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