Journal article Open Access

A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites

Felix Moran; Antonio Olmos; Leonidas Lotos; Lukas Predajňa; Nikolaos Katis; Miroslav Glasa; Varvara Maliogka; Ana B. Ruiz-Garcia


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    <subfield code="a">GPGV, Grapevine, Real-time RT-PCR, Quantification</subfield>
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    <subfield code="u">Centro de Proteccion Vegetal y Biotecnologia, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain</subfield>
    <subfield code="a">Antonio Olmos</subfield>
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    <subfield code="u">Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Plant Pathology Laboratory, Thessaloniki, Greece</subfield>
    <subfield code="a">Leonidas Lotos</subfield>
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    <subfield code="u">Institute of Virology, Biomedical Research Centre, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia</subfield>
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    <subfield code="u">Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Plant Pathology Laboratory, Thessaloniki, Greece</subfield>
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    <subfield code="u">Institute of Virology, Biomedical Research Centre, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia</subfield>
    <subfield code="a">Miroslav Glasa</subfield>
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    <subfield code="u">Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture, Plant Pathology Laboratory, Thessaloniki, Greece</subfield>
    <subfield code="a">Varvara Maliogka</subfield>
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    <subfield code="u">Centro de Proteccion Vegetal y Biotecnologia, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain</subfield>
    <subfield code="a">Ana B. Ruiz-Garcia</subfield>
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    <subfield code="c">2018-05-15</subfield>
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    <subfield code="u">Centro de Proteccion Vegetal y Biotecnologia, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain</subfield>
    <subfield code="a">Felix Moran</subfield>
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    <subfield code="a">A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites</subfield>
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    <subfield code="a">&lt;p&gt;&lt;em&gt;Grapevine Pinot gris virus&lt;/em&gt; (GPGV) is a widely distributed grapevine pathogen that has been associated to the grapevine leaf mottling and deformation disease. With the aim of better understanding the disease epidemiology and providing efficient control strategies a specific and quantitative duplex TaqMan real-time RT-PCR assay has been developed. This method has allowed reliable quantitation of the GPGV titer ranging from 30 up to 3 x 10&lt;sup&gt;8&lt;/sup&gt; transcript copies, with a detection limit of 70 viral copies in plant material. The assay targets a grapevine internal control that reduces the occurrence of false negative results, thus increasing the diagnostic sensitivity of the technique. Viral isolates both associated and non-associated to symptoms from Greece, Slovakia and Spain have been successfully detected. The method has also been applied to the absolute quantitation of GPGV in its putative transmission vector &lt;em&gt;Colomerus vitis&lt;/em&gt;. Moreover, the viral titer present in single mites has been determined. In addition, in the current study a new polymorphism in the GPGV genome responsible for a shorter movement protein has been found. A phylogenetic study based on this genomic region has shown a high variability among Spanish isolates and points to a different evolutionary origin of this new polymorphism. The methodology here developed opens new possibilities for basic and epidemiological studies as well as for the establishment of efficient control strategies.&lt;/p&gt;</subfield>
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