Real-time assay for quantitative detection of non-persistently transmitted Plum pox virus RNA targets in single aphids

Olmos, Antonio; Bertolini, Edson; Gil, Maite; Cambra, M.

doi:10.1016/j.jviromet.2005.05.011

Cited by 116 publications

(80 citation statements)

References 24 publications

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“…Real-time PCR is an adaptation of the traditional PCR protocol that allows rapid detection of target-specific amplicons without post-PCR electrophoresis. This assay was successfully developed for PPV diagnosis and characterization (Schneider et al, 2004;Olmos et al, 2005;. The use of molecular techniques, availability of full-length and partial sequences and phylogenetic analysis have enabled precise and reliable strain classification.…”

Section: Plum Pox Virus Strain Variabilitymentioning

confidence: 99%

Plum pox virus strains: Diversity and geographical distribution in Serbia

Jevremović

Paunović

2014

Pesticidi i fitomedicina

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Plum pox virus (PPV) is the causal agent of Sharka disease. Since its discovery, Sharka has been considered as a calamity in plum orchards. PPV is present worldwide in many Prunus species, causing great economic losses. In highly susceptible plum varieties, such as Pozegaca, PPV causes a premature fruit drop and reduces fruit quality, which leads to total yield loss. Eight PPV strains (PPV-M, PPV-D, PPV-EA, PPV-C, PPV-Rec, PPV-W, PPV-T and PPVCR) have been recognized so far. Three major strains (PPV-M, PPV-D and PPV-Rec) are the most widely dispersed and occur frequently in many European countries. Other strains are of minor importance due to their limited host preferences or geographic distribution. So far, all three major strains have been identified in Serbia. In this paper, we provide a comprehensive overview of the research into Plum pox virus variability in Serbia. [Projekat Ministarstva nauke Republike Srbije, br TR-20013A i br. TR-31064. This study was partially supported by the ECONET grant no. 10159PL, the FP7 project SharCo]

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Section: Plum Pox Virus Strain Variabilitymentioning

confidence: 99%

Plum pox virus strains: Diversity and geographical distribution in Serbia

Jevremović

Paunović

2014

Pesticidi i fitomedicina

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“…pmol of ssRNA = lg (of ssRNA) 9 (10 6 pg/1 lg) 9 (1 pmol/340 pg) 9 (1/Nb). Avogadro constant was used to estimate the number of transcripts (6.023 9 10 23 molecules/mol) [14].…”

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confidence: 99%

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Gadiou

Kundu

2012

Indian J. Virol.

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A SYBR Green Ò -based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plantexpressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and bactin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.The fluorescence-based quantitative real-time PCR (qPCR) has been used to detect and measure nucleic acids of a wide range of plant viruses with RNA and DNA genome [13,16,17]. Two main qPCR based techniques: relative and absolute quantification have emerged from various studies on virus detection and quantification of virus titer. The ''absolute quantification'' with known standard is the most commonly used for plant virus quantification. On the other hand, relative quantification uses a comparative quantification with internal reference endogenous housekeeping control genes [1]. The relative quantification determines the changes in RNA levels of a gene across multiple samples and expresses it relative to the levels of an internal control RNA. Therefore, relative quantification does not require standards with known concentrations [15]. Relative quantification employing reference internal control genes was used for several plant virus detection and quantification studies [5,6]. A comparative study showed that the relative quantification assay may be as accurate as absolute quantification when appropriate housekeeping genes are used and normalized with target genes [6,10]. In this paper we have chosen endogenous housekeeping genes for quantification of viruses in apple trees in a relative quantification system. Two apple tree infecting viruses, Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) are selected. ASGV and ApMV belong to the most frequently emerged pathogens encounters in pome fruit trees. Several molecular-based methods have been elaborated to perform routine detection [7,8,11,12]. However, a highly sensitive real-time based detection and quantification assay for these viruses is missing so far. We described here a SYBER green based relative quantification assay employing appropriate endogenous reference genes as internal control.A total of 39 samples coming from different apple cultivars grown in vitro culture (i.e. Idared, Champion, Fragrance) healthy (14 plants) and infected (25 plants) have been used to validate the reference genes as well as the target genes. The leaves of plant material were grinded in liquid nitrogen and the total RNA ...

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“…2007) y es similar a la sensibilidad obtenida con otros virus (Fabre et al, 2003;Korimbocus et al, 2002;Picó et al, 2005;Roberts et al, 2000). La detección de Barley yellow dwarf virus o de Plum pox virus mediante q-RT-PCR fue 10 3 veces más sensible que con ELISA, (Fabre et al, 2003;Olmos et al, 2005 Utilizando q-RT-PCR se observó que las diferencias de acumulación de cada aislado en los distintos huéspedes, o de distintos aislados en un mismo huésped, en la primera brotación, se mantenían a los 9 meses p.i. y que la carga viral no estaba relacionada con la virulencia del aislado en cada huésped.…”

Section: Discussionunclassified

Estudio de la interacción entre naranjo amargo y el virus de la tristeza de los cítricos

Serra¹

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Real-time assay for quantitative detection of non-persistently transmitted Plum pox virus RNA targets in single aphids

Cited by 116 publications

References 24 publications

Plum pox virus strains: Diversity and geographical distribution in Serbia

Plum pox virus strains: Diversity and geographical distribution in Serbia

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Estudio de la interacción entre naranjo amargo y el virus de la tristeza de los cítricos

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