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 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 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 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 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 Expression of 5’-deiodinase in various thyroid tumors

2004 ◽  
Vol 50 (3) ◽  
pp. 34-37
Author(s):  
G. M. Artykbaeva ◽  
Ya. Kh Turakulov
Keyword(s):  
Polymerase Chain Reaction ◽  
Thyroid Carcinoma ◽  
Gel Electrophoresis ◽  
Human Thyroid ◽  
Thyroid Tumors ◽  
Great Role ◽  
Chain Reaction ◽  
Pcr Products ◽  
Important Pathway ◽  
Polymerase Chain

5'-deiodination is an important pathway for the metabolism of T4 by determining its great role as a prohormone for T3. T4 is metabolized by two 5'-deiodinases: DI and DII which are encoded by different genes, regulated by different modes and expressed in various tissues. The aim of the present study was to examine DI and DII expression in health and in various human thyroid tumors. Eighteen thyroid specimens (10 from tumors and 8 from the intact thyroid tissues surrounding the nodes) were used. RNA isolated from the tissues by reserve transcription, followed by polymerase chain reaction (PCR) were transcribed to cDNA. Gel electrophoresis showed the bands corresponding to 177 b. p for DI and 796 b. p for DII. The experiments demonstrated that there were genes of both isoenzymes of 5 ’deiodinases in thyroid carcinoma tissues. Gel electrophoresis indicated the different expression of PCR products in different thyroid tumors for DI and DII at the level of mRNA.

scholarly journals Detection of Acanthamoeba spp. in two major water reservoirs in the Philippines

2020 ◽  
Vol 18 (2) ◽  
pp. 118-126 ◽  
Author(s):  
Giovanni Milanez ◽  
Frederick Masangkay ◽  
Frieda Hapan ◽  
Thea Bencito ◽  
Marcus Lopez ◽  
...  
Keyword(s):  
Water Samples ◽  
Gel Electrophoresis ◽  
Water Security ◽  
The Philippines ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Water Reservoirs ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain

Abstract Water reservoirs are important manmade structures providing water security to deliver clean and safe water for drinking and other purposes to the community. Eighty water samples were collected from Magat and Ipo water reservoirs using purposive sampling between November 2018 and January 2019. Water samples were collected in sterile containers for testing. The samples were cultured in non-nutrient agar and lawned with Escherichia coli and incubated at 33 °C. Twelve out of the 80 (15%) water samples were positive for amoebic growth. Light and scanning electron microscopy (SEM) revealed double-walled cystic stages and were initially identified as Acanthamoeba spp. based on morphological characteristic in reference to Page's established criteria. Their extracted DNAs were used in polymerase chain reaction using JDP1 and JDP2 primers and confirmed the presence of Acanthamoeba DNA in agarose gel electrophoresis. Aligned sequences from PCR products were deposited in GenBank under accession numbers MK886460, MK909919, MK905437, MK910997, MK911021 and MK886514. The presence of potentially pathogenic Acanthamoeba spp. in water reservoirs is considered a potential risk for public health, requiring appropriate processing of water in treatment plants.

scholarly journals Detection of Canine Parvovirus in Baghdad city by PCR technique

2012 ◽  
Vol 36 (0E) ◽  
pp. 95-98
Author(s):  
Ahmed F. Ahmed
Keyword(s):  
Gel Electrophoresis ◽  
Canine Parvovirus ◽  
Chain Reaction ◽  
Viral Dna ◽  
Fatal Disease ◽  
The Third ◽  
Mutant Strains ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Hemorrhagic Enteritis

Canine parvovirus 2 (CPV2) is a highly contagious and fatal disease of dogs, causingacute hemorrhagic enteritis and myocarditis. In this study different mutant strains of the viruswere characterized by polymerase chain reaction (PCR).The fecal samples from infected dogssuspected for CPV2 infection were collected in a suitable medium. The viral DNA from fecalsamples was extracted using specific kits, PCR were carried out with five different primer,pCPV-2ab and pCPV-2b, to distinguish the strain prevalent in field condition. The primerpCPV-2ab recognized both variant CPV-2a and CPV-2b, whereas the primer pCPV-2brecognized only the variant CPV-2b, using the third primer pCPV to recognize the residualbase pair, enabling the differentiation of CPV-2a variant from CPV-2b in field isolates. Thedifferent PCR products were further analyzed by using gel electrophoresis.

scholarly journals Upregulation of the miRNA-155, miRNA-210, and miRNA-20b in psoriasis patients and their relation to IL-17

2020 ◽  
Vol 34 ◽  
pp. 205873842093374 ◽  
Author(s):  
Mohamed El-Komy ◽  
Iman Amin ◽  
Marwa Safwat El-Hawary ◽  
Dina Saadi ◽  
Olfat Shaker
Keyword(s):  
Serum Levels ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epigenetic Mechanisms ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Lesional Skin ◽  
Immune Mediated ◽  
Elisa Technique ◽  
Polymerase Chain ◽  
The Relationship

Psoriasis is an immune-mediated disease, with genetic background and triggering environmental factors; however, several gaps are still present in understanding the intertwined relationship between these elements. Epigenetic mechanisms, including microRNAs (miRNAs), play an important role in the pathogenesis of psoriasis. The relationship between interleukin (IL)-17, a key cytokine in psoriasis, and these epigenetic mechanisms still needs to be elucidated. This study aimed at assessing the expression of miRNA-155, miRNA-210, and miRNA-20b in skin and sera of psoriasis patients in relation to IL-17 levels. For 20 psoriasis patients and 20 matching controls, the expression of miRNA-155, miRNA-210, and miRNA-20b was assessed using real-time polymerase chain reaction (RT-PCR), whereas IL-17/IL-17A levels were measured using quantitative enzyme-linked immunosorbent assay (ELISA) technique. MiRNA-155 expression was significantly higher in lesional skin compared to controls ( P = 0.001). MiRNA-210 expression was significantly higher in both, lesional skin ( P = 0.010) and sera of patients ( P = 0.001) in comparison with controls. A statistically significant positive correlation was found between serum miRNA-210 expression and serum levels of IL-17/IL-17A ( P = 0.010, rs = 0.562). MiRNA-20b lesional and non-lesional expression was significantly higher than controls ( P < 0.001; P = 0.018). In conclusion, the expression of miRNA-155, miRNA-210, and miRNA-20b is exaggerated in psoriasis and they may be involved in disease pathogenesis. A possible relationship between miRNA-210 and IL-17 may be suggested; however, further studies are still needed to verify this relation.

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