2010
DOI: 10.1094/phyto-04-10-0102
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Development and Application of a Multiplex Reverse-Transcription Polymerase Chain Reaction Assay for Screening a Global Collection of Citrus tristeza virus Isolates

Abstract: The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex… Show more

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Cited by 46 publications
(38 citation statements)
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“…Multiplex RT-PCR assay has been developed successfully to detect and differentiate closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus [7], each of the major Potato virus Y strains and strain mixtures [4346]. CMV subgroups and tobamoviruses infecting tomato [17], genotypes A and B of Beet necrotic yellow vein virus [11], five genotypes of Citrus tristeza virus from 29 citrus-growing countries [29]. There are three BrYV genotypes (BrYV -A, -B, and -C) based on sequence comparisons and phylogenetic analysis [1, 4], however, the pathogenicity, distribution and host range of the three BrYV genotypes remained to be determined.…”
Section: Discussionmentioning
confidence: 99%
“…Multiplex RT-PCR assay has been developed successfully to detect and differentiate closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus [7], each of the major Potato virus Y strains and strain mixtures [4346]. CMV subgroups and tobamoviruses infecting tomato [17], genotypes A and B of Beet necrotic yellow vein virus [11], five genotypes of Citrus tristeza virus from 29 citrus-growing countries [29]. There are three BrYV genotypes (BrYV -A, -B, and -C) based on sequence comparisons and phylogenetic analysis [1, 4], however, the pathogenicity, distribution and host range of the three BrYV genotypes remained to be determined.…”
Section: Discussionmentioning
confidence: 99%
“…Complicating matters is a lack of consistency in choosing a region or regions to analyze, with CP, ORF1a/b, and sundry 3′ genes all being targeted in different assays (Hilf et al, 2005; Nolasco et al, 2009; Roy et al, 2010). Diagnosis with the coat protein alone is a historical legacy, as it is the most highly conserved and least variable of the 3′ genes with 93.4% nucleotide identity and 96.3% amino acid identity between isolates examined in this study.…”
Section: Discussionmentioning
confidence: 99%
“…The MMM genotype analysis of CTV was performed using reverse transcription polymerase chain reaction (RT-PCR) with specific primers for the genotypes T30, VT, T36, T3, and B165/T68, as previously described (Hilf et al, 2005; Roy and Brlansky, 2010; Roy et al, 2010) (Table 2). Total RNA from ~0.2 g of bark tissue was extracted using the Spectrum Plant Total RNA kit (Sigma, Saint Louis, Missouri, USA) according to the manufacturer's instructions and eluted in 30 μl of RNase-free water.…”
Section: Methodsmentioning
confidence: 99%
“…aTwo MMM methodologies were developed independently by Hilf et al (2005)(-H) and Roy and Brlansky (2010)(-R) and Roy et al (2010). CP-U: Coat protein universal primer was developed in this study and was used as positive internal control for CTV detection.…”
Section: Methodsmentioning
confidence: 99%
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