Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan®)

2008 ◽  
Vol 147 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Maria Saponari ◽  
Keremane Manjunath ◽  
Raymond K. Yokomi
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Quantitative Detection ◽  
Reverse Transcription Pcr ◽  
Tristeza Virus ◽  
Citrus Tristeza

scholarly journals Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR

2007 ◽  
Vol 120 (2) ◽  
pp. 177-188 ◽  
Author(s):  
Edson Bertolini ◽  
Aranzazu Moreno ◽  
Nieves Capote ◽  
Antonio Olmos ◽  
Ana de Luis ◽  
...  
Keyword(s):  
Real Time ◽  
Citrus Tristeza Virus ◽  
Quantitative Detection ◽  
Plant Tissues ◽  
Rt Pcr ◽  
Tristeza Virus ◽  
Citrus Tristeza

scholarly journals Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza virus by Real-Time Reverse Transcription-Polymerase Chain Reaction Assays

2010 ◽  
Vol 100 (4) ◽  
pp. 319-327 ◽  
Author(s):  
R. K. Yokomi ◽  
M. Saponari ◽  
P. J. Sieburth
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Sodium Dodecyl ◽  
Potassium Acetate ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

scholarly journals Discrimination Between Mild and Severe Citrus tristeza virus Isolates with a Rapid and Highly Specific Real-Time Reverse Transcription-Polymerase Chain Reaction Method Using TaqMan LNA Probes

2009 ◽  
Vol 99 (3) ◽  
pp. 307-315 ◽  
Author(s):  
S. Ruiz-Ruiz ◽  
P. Moreno ◽  
J. Guerri ◽  
S. Ambrós
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Dynamic Range ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 102 copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates.

scholarly journals Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

2005 ◽  
Vol 71 (9) ◽  
pp. 5624-5626 ◽  
Author(s):  
X. C. Shan ◽  
P. Wolffs ◽  
M. W. Griffiths
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Immunomagnetic Capture ◽  
Capture Method

ABSTRACT In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

scholarly journals Development and Application of a Multiplex Reverse-Transcription Polymerase Chain Reaction Assay for Screening a Global Collection of Citrus tristeza virus Isolates

2010 ◽  
Vol 100 (10) ◽  
pp. 1077-1088 ◽  
Author(s):  
Avijit Roy ◽  
G. Ananthakrishnan ◽  
John S. Hartung ◽  
R. H. Brlansky
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Phylogenetic Analyses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Variable Regions ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

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 (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

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