A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine

Dovas, Chrysostomos Ι.; Katis, N. I.

doi:10.1016/s0166-0934(02)00197-0

Cited by 74 publications

(38 citation statements)

References 14 publications

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“…According to the Eighth Report of the International Committee on Taxonomy of Viruses, Potexvirus species share more than ~72% identical nucleotide sequences between their polymerase genes , confirming CsCMV infection of some plants even after thermotherapy followed by meristematic-tip culture. This result is in agreement with the sensitivity levels reported for other PCR-based methods that proved more sensitive when the viral concentration was below the detection limit of ELISA (Dovas & Katis, 2003).…”

Section: Resultssupporting

confidence: 91%

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Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

Silva¹,

Carnelossi²,

Bijora³

et al. 2011

Trop. plant pathol.

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A virus survey conducted in the northwest region of Paraná, the main cassava-producing region of that state, showed Cassava common mosaic virus (CsCMV) to be widespread, infecting more than 90% of plants from all cassava cultivars. A CsCMV isolate was purified and used to raise a high-titer (1/1.000) polyclonal antiserum for indexing plants produced from meristem-tip culture, using PTA-ELISA. From an initial production of 110 plants of cultivar Olho Junto, 31 remained infected as indicated by PTA-ELISA. To improve the sensitivity of virus detection, an immunocapture-RT-PCR (IC-RT-PCR) protocol was established. Virus-specific IgG, purified and combined with a primer set for the genus Potexvirus, could readily detect CsCMV in cassava crude leaf extracts. IC-RT-PCR products of 750 bp were amplified from six out of 35 plants previously tested as virus-negative by PTA-ELISA. Sequence analysis of cloned IC-RT-PCR products confirmed they were part of the CsCMV replicase gene, indicating that the virus was still present after thermotherapy and meristem-tip culture. PTA-ELISA enabled initial screening of virus-positive cassava, reducing the number of plants to be further analyzed by IC-RT-PCR. Though CsCMV has been considered of minor importance, its widespread nature, as noticed in Paraná, indicates the need for adoption of effective control measures.

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Section: Resultssupporting

confidence: 91%

“…In an attempt to overcome this problem, RT-PCR has been widely used for detection of plant viruses (Dovas & Katis, 2003).…”

Section: Resultsmentioning

confidence: 99%

Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

Silva¹,

Carnelossi²,

Bijora³

et al. 2011

Trop. plant pathol.

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“…For the simultaneous amplification of viruses of the Foveavirus and Vitivirus genera, degenerate primer pairs dRW up1/dRW do2 and dRW nest1/dRW nest2, reported by Dovas and Katis [6] were used. Primers dRW up1/dRW do2 were used in RT and in the first round of PCR, and primers dRW nest1/dRW nest2 in the second round.…”

Section: Isolation Of Dsrnamentioning

confidence: 99%

“…To determine whether molecular variants of GVB were present in the GVB-infected plants LN953(CB) and LN327(H), the virus was amplified from dsRNA using RTnested PCR based on the degenerate primers of Dovas and Katis [6]. These primers probably amplify all molecular variants of GVB as they were designed for simultaneous amplification of all viruses of the Foveavirus and Vitivirus genera.…”

Section: Identification Of Gvb Variants Using Sscp Analysis Of Rt-pcrmentioning

confidence: 99%

Divergent molecular variants of Grapevine virus B (GVB) from corky bark (CB)-affected and CB-negative LN33 hybrid grapevines

Goszczynski

2010

Virus Genes

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Analysis of two Grapevine virus B (GVB)-infected LN33 hybrid grapevines revealed that a plant exhibiting clear symptoms of corky bark (CB) disease was infected with two molecular variants of the virus, whereas a plant exhibiting no disease symptoms was infected with only one variant. Sequence results indicated that the single variant in the CB-negative grapevine was also one of the two present in the CB-affected hybrid. Plant extracts from these two grapevines were used to successfully transmit the virus to N. benthamiana. After further cloning and sequencing, two clearly divergent variants were identified. Comparative molecular analysis of the variants, named here GVB 953-1 and GVB-H1, respectively, transmitted from CB-affected and consistently CB-negative plants, revealed short genomic regions, most of them highly divergent, that encoded amino acid sequences, containing significant amino acid substitutions altering the net charges of their respective proteins. Interestingly, a comparison of these variants to genome sequence data of GVB variants GVB Italy and GVB 94/971 available from the GenBank, revealed that these significant amino acid substitutions were the same for, and unique to, the variant pairs GVB 953-1/GVB Italy and GVB-H1/GVB 94/971. This despite the variants of each pair being otherwise clearly different at nucleotide and amino acid levels. In addition, both sets of variants differed substantially in their respective 3'-non-translated (3'NTR) regions. The relevance of these findings is discussed.

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“…The results involving the replicase gene, considered a conserved protein for ssRNA viruses (Dovas & Kátis, 2003a, b), demonstrate potential for detection of unknown viruses from the family Comoviridae. The possible molecular difference among the CPSMV isolates could, probably, be detected when the whole polyprotein was sequenced and analyzed (Dovas & Katis, 2003a;Maliogka et al, 2004). Amplified DNA fragments with estimated size of 593 bp from the CPSMV isolates were cloned and the fragments from the CPSMV-PE isolate showed a small difference in size ( Figure 1C), indicating a possible molecular difference in this virus isolate.…”

Section: Nicotiana Tabacum Ns/ (-) Ns/ (-) Ns/(-) Ns/ (-) Ns/ (-)mentioning

confidence: 99%

Biological, serological and molecular comparison between isolates of Cowpea severe mosaic virus

Camarço¹,

Nascimento²,

Andrade³

et al. 2009

Trop. plant pathol.

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Cowpea, Vigna unguiculata, can be affected by several diseases, and those caused by viruses are considered of great importance. Cowpea severe mosaic virus (CPSMV), which belongs to the family Comoviridae, genus Comovirus, stands out for its severity and degree of incidence. Its genetic variability can give rise to different strains around the world, including in Brazil. The objective of the present research was to establish a biological, serological and molecular comparison between five CPSMV isolates obtained from naturally infected plants in different States of the Northeast of Brazil. Isolates were obtained from cowpea: CPSMV-AL (State of Alagoas), CPSMV-CE (Ceará); CPSMV-MC (Piauí), CPSMV-PE (Pernambuco) and an isolate obtained from Crotalaria paulinea, in the State of Maranhão -CPSMV-CROT. A host range study evidenced biological differences among the virus isolates, especially in cowpea genotypes. The isolates were showed to be serologically and molecularly related by polymerase chain reaction �� PCR, using degenerated primers which amplified two conserved regions in the coat protein and in the replicase genes. Cloning and sequence of CPSMV-PE made possible its comparison with other CPSMV isolates and other virus species from the genus Comovirus. Keywords: Vigna unguiculata, Comovirus, CPSMV, molecular differentiation. RESUMO Comparação biológica, sorológica e molecular de isolados de Cowpea severe mosaic virusO feijão caupi, Vigna unguiculata, pode ser afetado por várias moléstias, entre as quais, destacam-se as viroses, cuja incidência e severidade variam, dependendo do hospedeiro, do vetor e da fonte do inóculo no campo. O vírus do mosaico severo do caupi (Cowpea severe mosaic virus, CPSMV) é o único vírus pertencente ao gênero Comovirus já identificado no Brasil infetando o feijão caupi. A presente pesquisa teve por objetivo estudar e comparar as características biológicas, sorológicas e moleculares de cinco isolados do CPSMV originários de diferentes estados do Nordeste: Isolados de feijão caupi em Alagoas (CPSMV-AL), no Ceará (CPSMV-CE); no Piauí (CPSMV-MC), em Pernambuco (CPSMV-PE) e o isolado obtido de Crotalaria paulinea no Maranhão (CPSMV-CROT). Os estudos de gama de hospedeiro evidenciaram diferenças biológicas entre os isolados, sobretudo em genótipos de caupi. As análise sorológicas em dupla difusão em agar confirmaram o forte relacionamento entre os isolados, e os estudos moleculares pela reação em cadeia da polimerase -PCR, utilizando oligonucleotídeos degenerados que amplificam seqüências conservadas dos genes do capsídeo e da replicase, não permitiram nenhuma diferenciação entre os isolados. A clonagem e o sequenciamento do CPSMV-PE possibilitaram sua comparação com outros isolados de CPSMV e outras espécies de vírus do gênero Comovirus.

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A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine

Cited by 74 publications

References 14 publications

Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

Divergent molecular variants of Grapevine virus B (GVB) from corky bark (CB)-affected and CB-negative LN33 hybrid grapevines

Biological, serological and molecular comparison between isolates of Cowpea severe mosaic virus

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