scholarly journals Extraction and molecular detection of viral dsRNA from different infected plants

2017 ◽  
Vol 1 ◽  
pp. 197
Author(s):  
Afsaneh Delpasand Khabbazi ◽  
Nemat Sokhandan Bashir ◽  
Saber Delpasand Khabbazi ◽  
Hakimeh Ighani
Keyword(s):  
Molecular Detection ◽  
Extraction Efficiency ◽  
Extraction Procedure ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Dsrna ◽  
Double Stranded Rna ◽  
Modified Method ◽  
Phenol Treatment ◽  
Polymerase Chain

Extraction of viral double stranded RNA (dsRNA) from infected plants is helpful in identification of the viruses involved in infection. To date, there have been several methods developed to isolate dsRNA; however, type of the plant and virus is determinative in extraction efficiency. In this study we extracted dsRNA from different woody and herbaceous plants through a modified method which reduces the costs and time of extraction procedure. This method is based on different affinity of nucleic acids for the cellulose CF-11 in1X STE (Sodium chloride Tris EDTA) buffer containing 16 % ethanol. There is no phenol treatment or mini columns used in the isolation procedure. Extracted dsRNAs were identified by ribonuclease treatment and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). We have applied the procedure on five different hosts representing Amaranthaceae, Vitaceae, Fabaceae and Rosaceae infected with four different viruses representing Secoviridae and Bromoviridae. 

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.

scholarly journals Molecular detection and characterisation of mumps virus in cerebrospinal fluid in a Gauteng laboratory

2016 ◽  
Vol 31 (1) ◽  
pp. 29-31
Author(s):  
Marieke Brauer ◽  
Marianne Wolfaardt ◽  
Lynne M. Webber ◽  
Maureen B. Taylor
Keyword(s):  
Polymerase Chain Reaction ◽  
Phylogenetic Analysis ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Reverse Transcription ◽  
Molecular Detection ◽  
Mumps Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The study aimed to determine the presence of mumps virus (MuV) in cerebrospinal fluid (CSF) specimens and to genetically characterise detected MuV strains. A real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the MuV F gene, and characterisation was performed by sequencing of the SH gene. Mumps virus was detected in 1.2% (3/260) of specimens. Phylogenetic analysis of one MuV strain revealed that it clustered with the Jeryl-Lynn and RIT4385 vaccine strains. As far as the authors could ascertain this is the first study to provide viral proof that these vaccine-like strains may be associated with aseptic meningitis.

Molecular Detection of Tobacco rattle virus in Bleeding Heart [Dicentra spectabilis (L.) Lem.] Growing in Alaska

2013 ◽  
Vol 14 (1) ◽  
pp. 57
Author(s):  
Nancy L. Robertson
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Detection ◽  
Tobacco Rattle Virus ◽  
Virus Transmission ◽  
Rt Pcr ◽  
Chain Reaction ◽  
South Central ◽  
Polymerase Chain ◽  
First Time ◽  
Dicentra Spectabilis

Tobacco rattle virus (TRV) was detected in bleeding heart from South Central Alaskan home gardens in 2010-11. TRV M-type and NM-type isolates were confirmed from these symptomatic bleeding heart plants by reverse transcription (RT)-PCR polymerase chain reaction, protein, serological, and virus transmission assays. RNA1 was sequenced from one of the bleeding heart M-type isolates, and the nucleotide identity ranged from 91% to 94% when compared with six TRV isolates from potato, spinach, and alstroemeria. This is the first detection of TRV from D. spectabilis in Alaska. It is also the first time that M- and NM-type isolates have been distinguished from bleeding heart plants. The significance of these findings is that even though TRV infected plants containing NM-type isolates probably will not be spread to other plants by its specific nematode vector; vegetative propagated roots from TRV infected plants of either type of isolates will continue to be a source of diseased plants to home gardeners. Accepted for publication 18 December 2012. Published 27 February 2013.

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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