scholarly journals Improved PCR Detection of Blackcurrant reversion virus in Ribes and Further Evidence that It Is the Causal Agent of Reversion Disease

Plant Disease ◽  
2002 ◽  
Vol 86 (12) ◽  
pp. 1333-1338 ◽  
Author(s):  
A. Teifion Jones ◽  
Wendy J. McGavin
Keyword(s):  
Causal Agent ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flower Bud ◽  
Annealing Temperatures ◽  
Reversion Virus ◽  
Polymerase Chain ◽  
Triton X ◽  
Red Currant

Within 5 years of mechanically inoculating blackcurrant cultivars with partially purified preparations of particles of Blackcurrant reversion virus (BRV), infected plants developed leaf and flower bud symptoms typical of reversion disease, demonstrating that BRV is the causal agent of this disease. To improve the erratic immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) detection of BRV in Ribes plants, various stepwise changes were made to the original protocol. Significant improvement in the reliability and sensitivity of BRV detection was made by extracting RNA from trapped BRV particles using Triton-X 100, the design of new primers with higher annealing temperatures, and the use of ‘Ready-to-go’ RT-PCR beads. These features, combined with other minor changes to the protocol, improved BRV detection in reverted blackcurrant plants from <50% to >90% but the reliability of BRV detection in red currant was always very much less and was possible only using nested PCR that was developed for this purpose.

scholarly journals Low incidence of peach latent mosaic viroid in peach mother blocks in Serbia

2014 ◽  
Vol 29 (2) ◽  
pp. 109-114
Author(s):  
Darko Jevremovic ◽  
Svetlana Paunovic
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Mosaic Disease ◽  
Causal Agent ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Low Incidence

Peach latent mosaic viroid (PLMVd) is the causal agent of peach latent mosaic disease that is common on peaches and nectarines worldwide. Most of the isolates do not cause any symptoms on the foliage and the disease may be latent for years. A survey to investigate the presence of PLMVd in selected peach mother blocks in 9 Serbian districts was carried out in 2011 through 2013. A total of 315 trees/samples originating from 43 mother blocks, representing 35 peach and nectarine varieties and 2 rootstocks, were tested by Reverse Transcription - Polymerase Chain Reaction (RT-PCR). PLMVd was detected in 13 samples (4.13%) belonging to 7 varieties and one vineyard peach rootstock. Infected samples were found in 7 mother blocks from 3 districts. Our results indicated a low incidence of PLMVd in the analyzed peach mother blocks.

scholarly journals Use of Degenerate Primers for Partial Sequencing and RT-PCR-Based Assays of Grapevine Leafroll-Associated Viruses 4 and 5

1998 ◽  
Vol 88 (11) ◽  
pp. 1238-1243 ◽  
Author(s):  
Geoffrey Routh ◽  
Yun-Ping Zhang ◽  
Pasquale Saldarelli ◽  
Adib Rowhani
Keyword(s):  
Heat Shock Protein 70 ◽  
Plasmid Vector ◽  
Pcr Primers ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Double Stranded Rna ◽  
Pcr Products ◽  
Polymerase Chain

Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV

1994 ◽  
Vol 72 (05) ◽  
pp. 762-769 ◽  
Author(s):  
Toshiro Takafuta ◽  
Kingo Fujirmura ◽  
Hironori Kawano ◽  
Masaaki Noda ◽  
Tetsuro Fujimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Specific Protein ◽  
Platelet Membrane ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flow Cytometric ◽  
Polymerase Chain ◽  
Megakaryocytic Cell

SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

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