Reverse transcription-duplex-polymerase chain reaction for simultaneous detection of Citrus tristeza virus and ‘Candidatus Liberibacter’ from citrus plants

2010 ◽  
Vol 117 (6) ◽  
pp. 241-243 ◽  
Author(s):  
Charith R. Adkar-Purushothama ◽  
Fabio Quaglino ◽  
Paola Casati ◽  
Piero A. Bianco
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Simultaneous Detection ◽  
Chain Reaction ◽  
Candidatus Liberibacter ◽  
Polymerase Chain ◽  
Duplex Polymerase Chain Reaction ◽  
Tristeza Virus ◽  
Citrus Tristeza

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.

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 Detection and Isolate Differentiation of Citrus tristeza virus in Infected Field Trees Based on Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
2004 ◽  
Vol 88 (6) ◽  
pp. 625-629 ◽  
Author(s):  
Zhipeng Huang ◽  
Phyllis A. Rundell ◽  
Xiong Guan ◽  
Charles A. Powell
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Sweet Orange ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

Reverse transcription-polymerase chain reaction (RT-PCR) was compared with enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) for detection of non-decline-inducing and decline-inducing isolates of Citrus tristeza virus (CTV) in 21 field sweet orange and grapefruit plants on sour orange rootstock in Fort Pierce, FL. Among these samples, seven, six, and eight were infected with decline-inducing, non-decline-inducing, and both decline-inducing and non-decline-inducing isolates of CTV, respectively. However, there was not a good correlation between field symptoms and detection of the decline-inducing isolate. The results confirmed that RT-PCR is not only able to detect and differentiate decline-inducing and non-decline-inducing isolates of CTV in Florida, but also can detect both isolate types in a single field sweet orange or grapefruit tree. For most samples, results from RT-PCR, ELISA, and DTBIA were the same. However, the 320-bp fragments produced only from decline-inducing isolates were amplified from two sweet orange and two grapefruit samples that did not react with decline-inducing CTV-specific monoclonal antibody MCA13 in ELISA or DTBIA, indicating that RT-PCR has a higher sensitivity than these immunological tests for field sweet orange or grapefruit samples. Thus, RT-PCR is a simple, rapid, and specific procedure for CTV identification applicable to both research and diagnostic needs.

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.

A Multiplex Reverse Transcription Polymerase Chain Reaction Method for the Detection of Foodborne Viruses†

1999 ◽  
Vol 62 (10) ◽  
pp. 1210-1214 ◽  
Author(s):  
SORAYA I. ROSENFIELD ◽  
LEE-ANN JAYKUS
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Simultaneous Detection ◽  
Polymerase Chain Reaction Method ◽  
Human Enterovirus ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, hepatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 bp, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were separated by agarose gel electrophoresis, and amplicon identity was confirmed by Southern transfer followed by DNA hybridization using nonradio-active, digoxigenin-labeled internal probes. When tested on mixed, purified virus suspensions, the multiplex method achieved detection limits of ≤1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for all viruses. With further streamlining efforts such as single tube amplification and liquid hybridization, multiplex PCR offers advantages over cell culture methodology and monoplex PCR because it allows for rapid and cost-effective detection of several human enteric viruses in a single reaction tube.

scholarly journals Reverse-Transcription Polymerase Chain Reaction Detection of Citrus Tristeza Virus in Aphids

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1066-1069 ◽  
Author(s):  
Prem Mehta ◽  
R. H. Brlansky ◽  
S. Gowda ◽  
R. K. Yokomi
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Citrus Tristeza Virus ◽  
Protein Gene ◽  
Aphid Species ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus

A rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection of citrus tristeza virus (CTV) in three aphid species. Seven CTV isolates from a worldwide isolate collection were used for aphid acquisition feeding by three aphid species. These included the most efficient CTV vector, the brown citrus aphid, Toxoptera citricida; the melon aphid, Aphis gossypii; and the green peach aphid, Myzus persicae, a non-vector for CTV. A short procedure for nucleic acid extraction from single or groups of aphids was developed. Nucleic acid extracts from 1, 3, 5, and 10 aphids with acquisition-access periods of 24 and 48 h were reverse transcribed and amplified using primers for the coat protein gene of the Florida B3 (T-36) isolate of CTV. PCR-amplified fragments of approximately 670 bp were obtained from all the isolates tested and the amplified product from the aphids fed on citrus infected with isolate B3 was confirmed as the CTV coat protein gene by digesting with various restriction enzymes. This technique will be useful in investigations of CTV-vector-plant interactions and CTV epidemiology.

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