scholarly journals Immunolocation of Aquaporin 8 in Human Cataractous Lenticular Epithelial Cells

2017 ◽  
Vol 2 (3) ◽  
pp. 1-5 ◽  
Author(s):  
Rijo Hayashi ◽  
Shimmin Hayashi ◽  
Kazunori Fukuda ◽  
Miki Sakai ◽  
Shigeki Machida
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Immunohistochemical Staining ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Anterior Capsule ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Anterior Capsulotomy ◽  
Density Results

Purpose/Aim: Aquaporin 8 (AQP8) is a diffusion facilitator of hydrogen peroxide (H2O2) through cell membranes. The purpose of this study was to confirm and localize AQP8 in human lenticular epithelial cells (LECs). Materials and Methods: Lenticular anterior capsule samples, including LECs, were collected during cataract surgery of cataract patients after informed consent. The localization of AQP8 was detected by immunohistochemical staining using an antibody to AQP8. Real-time polymerase chain reaction (RT-PCR) was also used to determine the AQP8 mRNA expression levels. The PCR products were analyzed by gel electrophoresis following analyses of band density. Results: Immunohistochemical staining showed AQP8 was distributed throughout the whole area of the anterior capsulotomy. AQP8 labeling was observed surrounding and within the cytoplasm of LECs. RT-PCR and gel electrophoresis also revealed the presence of AQP8 mRNA in the lenticular anterior capsule. The results of immunohistochemical staining were comparable to those of RT-PCR and gel electrophoresis. Conclusions: The results of this study indicate the distribution of AQP8 in human LECs. This is the first investigation confirming the presence of AQP8 in human LECs.

scholarly journals Rapid Detection and Quantification of Rhynchosporium secalis in Barley Using a Polymerase Chain Reaction

2011 ◽  
Vol 42 (No. 3) ◽  
pp. 111-114
Author(s):  
J. Gubiš ◽  
M. Hudcovicová ◽  
M. Gubišová
Keyword(s):  
Gel Electrophoresis ◽  
Pcr Primers ◽  
Rhynchosporium Secalis ◽  
Agarose Gel Electrophoresis ◽  
Artificial Infection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Green Approach ◽  
Total Dna ◽  
Polymerase Chain

PCR primers for diagnosis of Rhynchosporium secalis in seed samples of barley were developed. For the quantification of the pathogen in seed samples a real-time PCR with SYBR Green approach was used. Amounts from 1.8 to 419.1 pg of R. secalis DNA per 100 ng of total DNA were detected in 18 samples of barley seeds contaminated by R. secalis in field conditions. The correctness of this quantitative analysis was checked using an artificial infection of seeds with 1, 2, 5 and 20% level of infection by R. secalis. The level of contamination of artificially infected samples decreased with a lowering amount of added seed powder contaminated by the pathogen, the correlation coefficient for this analysis was 0.98. While the primer pair used in these analyses shows cross-reactions with other pathogens (P. teres, Drechslera tritici-repentis, F. culmorum and F. poe), it is recommended to check the products of RT-PCR by agarose-gel electrophoresis, in which these pathogens are easily distinguishable from R. secalis by different lengths of the amplified fragments.  

scholarly journals Diversity of Ampeloviruses in Mealybug and Soft Scale Vectors and in Grapevine Hosts from Leafroll-Affected Vineyards

2009 ◽  
Vol 99 (10) ◽  
pp. 1177-1184 ◽  
Author(s):  
M. Fuchs ◽  
P. Marsella-Herrick ◽  
G. M. Loeb ◽  
T. E. Martinson ◽  
H. C. Hoch
Keyword(s):  
New York ◽  
Heat Shock Protein 70 ◽  
Host Plants ◽  
Protein Gene ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Pseudococcus Maritimus

The occurrence and diversity of Grapevine leafroll-associated virus 1 (GLRaV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3) in the soft scales Parthenolecanium corni and Pulvinaria innumerabilis and in the mealybug Pseudococcus maritimus was determined in leafroll-affected vineyards in the Finger Lakes region of New York. Groups of 1 to 4 specimens were collected under loose grapevine bark and tested by reverse-transcription polymerase chain reaction (RT-PCR) for segments of the second diverged copy of the GLRaV-1 coat protein gene or GLRaV-3 heat-shock protein 70-homologue gene. Virus-specific RT-PCR products were amplified from immature insect vectors and adult mealybugs. Single viral amplicons were obtained mostly from immature vectors (35%, 30 of 85) and dual viral amplicons from immature (16%, 10 of 61) and adult (100%, 14 of 14) mealybugs, including individuals. These observations suggested a simultaneous uptake of GLRaV-1 and GLRaV-3 by individual mealybugs. Furthermore, a comparative nucleotide sequence analysis of viral amplicons from soft scales, mealybugs, and grapevines from which vectors were collected showed identical or highly similar haplotypes, indicating that uptake of GLRaV-1 and GLRaV-3 likely occurred by direct feeding of vectors on their host plants.

scholarly journals Applicability of Three Alternative Instruments for Food Authenticity Analysis:GMO Identification

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
A. Burrell ◽  
C. Foy ◽  
M. Burns
Keyword(s):  
Polymerase Chain Reaction ◽  
Gel Electrophoresis ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Fitness For Purpose ◽  
Chain Reaction ◽  
Food Authenticity ◽  
Pcr Products ◽  
Polymerase Chain

Ensuring foods are correctly labelled for ingredients derived from genetically modified organisms (GMOs) is an issue facing manufacturers, retailers, and enforcement agencies. DNA approaches for the determination of food authenticitys often use the polymerase chain reaction (PCR), and PCR products can be detected using capillary or gel electrophoresis. This study examines the fitness for purpose of the application of three laboratory electrophoresis instruments (Agilent Bioanalyzer 2100, Lab901 TapeStation, and Shimadzu MCE-202 MultiNA) for the detection of GMOs using PCR based on a previously validated protocol. Whilst minor differences in the performance characteristics of bias and precision were observed, all three instruments demonstrated their applicability in using this protocol for screening of GMO ingredients.

scholarly journals Development of PCR-ELISA for Detection and Differentiation of Didymella bryoniae from Related Phoma species

Plant Disease ◽  
2002 ◽  
Vol 86 (7) ◽  
pp. 710-716 ◽  
Author(s):  
B. M. Somai ◽  
A. P. Keinath ◽  
R. A. Dean
Keyword(s):  
Gel Electrophoresis ◽  
Sulfonic Acid ◽  
Pcr Primers ◽  
Enzyme Linked Immunosorbent Assay ◽  
Didymella Bryoniae ◽  
Chain Reaction ◽  
Pcr Products ◽  
Elisa Technique ◽  
Polymerase Chain ◽  
Biotin Label

The causal agent of gummy stem blight, Didymella bryoniae, often is isolated from infected cucurbits together with other Phoma spp. Polymerase chain reaction (PCR) primers specific to D. bryoniae and Phoma were used to develop and evaluate a microtiter-based PCR-enzyme-linked immunosorbent assay (ELISA) technique. Primers were modified by addition of a fluorescein and a biotin label to the 5′ ends of the forward and reverse primers, respectively. After amplification, PCR products were detected in an ELISA using horseradish peroxidase-conjugated antifluorescein antibody and three substrates that yielded three colored products, one for each fungal group. The most sensitive substrate (highest signal:noise ratio) was 2,2′ -azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid]. PCR-ELISA successfully detected 45 of 46 D. bryoniae and all 13 Phoma isolates that were used. Results were comparable to those obtained with gel electrophoresis. Only one D. bryoniae isolate could not be detected with PCR-ELISA; this isolate also produced a fragment larger than other D. bryoniae isolates on agarose gels. PCR-ELISA was used successfully on crude extracts of “blind” fungal samples and identified seven of seven isolates as D. bryoniae or Phoma. Although less sensitive than gel electrophoresis, PCR-ELISA was a highly specific, yet simple, rapid and convenient assay for detection of D. bryoniae and Phoma sp.

scholarly journals Quantitative real-time RT-PCR – a perspective

2005 ◽  
Vol 34 (3) ◽  
pp. 597-601 ◽  
Author(s):  
S A Bustin ◽  
V Benes ◽  
T Nolan ◽  
M W Pfaffl
Keyword(s):  
Real Time ◽  
Gel Electrophoresis ◽  
Southern Blotting ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fluorescent Reporter ◽  
Specificity And Sensitivity ◽  
Homogeneous Assay ◽  
Polymerase Chain

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

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 Polyvalent Degenerate Oligonucleotides Reverse Transcription-Polymerase Chain Reaction: A Polyvalent Detection and Characterization Tool for Trichoviruses, Capilloviruses, and Foveaviruses

2005 ◽  
Vol 95 (6) ◽  
pp. 617-625 ◽  
Author(s):  
Xavier Foissac ◽  
Laurence Svanella-Dumas ◽  
Pascal Gentit ◽  
Marie-Josée Dulucq ◽  
Armelle Marais ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Unknown Etiology ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Sequence Comparisons ◽  
Pcr Products ◽  
Chlorotic Leaf ◽  
Polymerase Chain

A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

Real-time reverse transcription-polymerase chain reaction for detection of SYT-SSX translocation in synovial sarcoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 9553-9553
Author(s):  
J. Liu ◽  
K. Qu ◽  
C. Chai ◽  
H. Li ◽  
A. Sferruzza ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Synovial Sarcoma ◽  
Internal Control ◽  
Reverse Transcription ◽  
Gel Electrophoresis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

9553 Background: Synovial sarcoma is the most common non-rhabdomyosarcomatous soft tissue sarcoma in children and adolescents. A specific translocation, t(X;18), induces fusion of the SYT gene on chromosome 18 to an SSX gene on chromosome X. The resulting fusion gene consists of at least 2 subtypes with different breakpoints: SYT-SSX1(X;18)(p11.23;q11.2) and SYT-SSX2 (X;18)(p11.21;q11.2). Because t(X;18) transcripts occur in >90% of synovial sarcoma subtypes, this marker may be useful for diagnosis. We evaluated the accuracy of a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of the primary SYT-SSX fusion transcript types in formalin-fixed, paraffin-embedded (FFPE) tissues and frozen tissues. Methods: 17 tumors (7 synovial sarcomas, 4 Ewing’s sarcomas, 5 rhabdomyosarcomas, 1 small round blue-cell tumor), 4 normal tissues, and 4 control samples were tested for SYT-SSX translocations using real-time RT-PCR. Results were compared to those obtained with gel electrophoresis detection of amplified transcripts; discrepant results were confirmed by sequencing. Results: Concordance between real time RT-PCR and gel electrophoresis was 100% (25/25) for internal control genes and SYT-SSX1, and 92% (23/25) for SYT-SSX2. Of the 2 samples with discordant SYT-SSX2 results, 1 was positive by real-time RT-PCR but not gel electrophoresis and 1 was positive by electrophoresis but not real-time RT-PCR; in both cases, DNA sequencing confirmed the real-time RT-PCR results. The minimum percentage of tumor to normal cells required for detection of SYT-SSX fusion transcripts by real-time RT-PCR was 6.25%. Conclusions: This real-time RT-PCR assay appears to provide greater accuracy than gel electrophoresis for identification of SYT-SSX translocation and fusion types. No significant financial relationships to disclose.

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