scholarly journals Enteroviruses and Bacteriophages in Bathing Waters

2005 ◽  
Vol 71 (11) ◽  
pp. 6838-6844 ◽  
Author(s):  
Laura Mocé-Llivina ◽  
Francisco Lucena ◽  
Juan Jofre
Keyword(s):  
Reverse Transcription ◽  
Bacteroides Thetaiotaomicron ◽  
Traditional Methods ◽  
Bacterial Indicators ◽  
Somatic Coliphages ◽  
Viral Genomes ◽  
Reverse Transcription Pcr ◽  
Bathing Waters ◽  
Good Potential

ABSTRACT A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages were also tested. Cultivable enteroviruses were detected in 55% of the samples, most of which complied with bacteriological criteria. In contrast, viral genomes were only detected in 20% of the samples by reverse transcription-PCR. Somatic coliphages outnumbered all other indicators. F-specific RNA phages were detected in only 15% of the samples, whereas phages infecting Bacteroides thetaiotaomicron were detected in 70% of samples. A numerical relationship between the numbers of enteroviruses and the numbers of enterococci and somatic coliphages was observed. In situ inactivation experiments showed that viruses persisted significantly longer than the bacterial indicators. Only somatic coliphages and bacteriophages infecting Bacteroides persisted longer than the viruses. These results explain the numbers of enteroviruses and indicators in bathing waters attending the numbers usually found in sewage in the area. Somatic coliphages show a very good potential to predict the risk of viruses being present in bathing waters.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

scholarly journals NewIn SituCapture Quantitative (Real-Time) Reverse Transcription-PCR Method as an Alternative Approach for Determining Inactivation of Tulane Virus

2014 ◽  
Vol 80 (7) ◽  
pp. 2120-2124 ◽  
Author(s):  
Dapeng Wang ◽  
Shuxia Xu ◽  
David Yang ◽  
Glenn M. Young ◽  
Peng Tian
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Viral Genome ◽  
Viral Infectivity ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Alternative Approach ◽  
Tulane Virus ◽  
Pcr Method

ABSTRACTHuman noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome byin situqRT-PCR. We first demonstrated that thisin situcapture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viral RNA or to transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.

scholarly journals In situ reverse transcription–PCR in plant tissues

1998 ◽  
Vol 3 (1) ◽  
pp. 1-4
Author(s):  
Laura Bombelli ◽  
Luisa Doneda ◽  
Chiara Tonelli ◽  
Silvana Dolfini
Keyword(s):  
Reverse Transcription ◽  
Plant Tissues ◽  
Reverse Transcription Pcr

scholarly journals In Situ Reverse Transcription-PCR for Monitoring Gene Expression in Individual Methanosarcina mazei S-6 Cells

2000 ◽  
Vol 66 (5) ◽  
pp. 1796-1800 ◽  
Author(s):  
Marianne Lange ◽  
Tim Tolker-Nielsen ◽  
Søren Molin ◽  
Birgitte K. Ahring
Keyword(s):  
Heat Shock ◽  
Reverse Transcription ◽  
Detection System ◽  
Permeabilized Cells ◽  
Methanosarcina Mazei ◽  
Reverse Transcription Pcr ◽  
Fast Red ◽  
Monitoring Gene Expression ◽  
Cellular Dna

ABSTRACT An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress genednaK. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate–Fast Red detection system and binding of anti-digoxigenin–alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the dnaK transcript.

Altered Signaling and Regulatory Mechanisms of Apoptosis in Focal and Segmental Glomerulosclerosis

2001 ◽  
Vol 12 (7) ◽  
pp. 1422-1433 ◽  
Author(s):  
WANSHENG WANG ◽  
ALEX TZANIDIS ◽  
MAJA DIVJAK ◽  
NAPIER MAURICE THOMSON ◽  
ALICIA NOEMI STEIN-OAKLEY
Keyword(s):  
Renal Function ◽  
Reverse Transcription ◽  
Fas Ligand ◽  
Regulatory Mechanisms ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tubulointerstitial Injury ◽  
Focal And Segmental Glomerulosclerosis ◽  
Reverse Transcription Pcr ◽  
Segmental Glomerulosclerosis

Abstract. The purpose of this study was to investigate signaling and regulatory mechanisms of apoptosis in a model of focal and segmental glomerulosclerosis. Sprague-Dawley rats received two doses of puromycin aminonucleoside (PAN) (day 0 and week 3) and a uninephrectomy (PAN model). Apoptosis was detected with the use of the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling technique. Bax, Bcl-2, Fas, and Fas ligand expression was analyzed by competitive reverse transcription-PCR. Bax, Bcl-2, and Fas mRNA were localized by in situ hybridization. Renal function was transiently impaired after the first PAN dose. After the second PAN dose, further progressive renal impairment, tubular atrophy, interstitial fibrosis, and glomerulosclerosis were evident. Eighteen percent of PAN samples demonstrated up to 4 apoptotic cells/50 glomeruli, compared with 7% of sham controls (not significant). No consistent significant change in glomerular Bax, Bcl-2, Fas, and Fas ligand mRNA was evident by reverse transcription-PCR, although focal increases in glomerular Bcl-2 mRNA were demonstrated by in situ hybridization. In the tubulointerstitium, apoptosis was increased from weeks 1 to 12 (P < 0.01 PAN versus sham), correlated to renal function and tubulointerstitial injury (P < 0.01). Total renal Bax, Fas, and Fas ligand mRNA were upregulated in the PAN model, peaking at week 17 (P < 0.01 versus sham), whereas Bcl-2 mRNA was not significantly different in PAN versus sham controls. In situ hybridization in the PAN model demonstrated prominent Bax mRNA in dilated tubules and infiltrating leukocytes. Fas mRNA signal was localized to tubular epithelial cells and leukocytes. The results suggest that altered apoptotic signaling and regulatory mechanisms contribute to the tubulointerstitial injury in this model.

scholarly journals Prolactin Receptor Messenger Ribonucleic Acid in Normal and Neoplastic Human Pituitary Tissues1

1997 ◽  
Vol 82 (3) ◽  
pp. 963-968
Author(s):  
Long Jin ◽  
Xiang Qian ◽  
Elzbieta Kulig ◽  
Bernard W. Scheithauer ◽  
Rocio Calle-Rodrigue ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Reverse Transcription ◽  
Ribonucleic Acid ◽  
Prolactin Receptor ◽  
Pituitary Surgery ◽  
Specific Cell ◽  
Human Pituitary ◽  
Messenger Ribonucleic Acid ◽  
Reverse Transcription Pcr

Abstract We examined the specific cell types in normal human pituitaries that expressed PRL receptor (PRL-R) messenger ribonucleic acid (mRNA) by combined in situ hybridization and immunohistochemistry. The distribution of PRL-R mRNA in 28 pituitary adenomas was examined by in situ hybridization and reverse transcription-PCR in 12 cases of adenomas. In another set of experiments, 34 PRL adenomas from men, women, and bromocriptine-treated patients were analyzed for PRL-R by in situ hybridization. In the normal pituitary, PRL- and LH-producing cells had significantly more mean grain counts per cell and higher percentages of cells positive for PRL-R than GH and TSH cells. PRL-R mRNA was present in all groups of adenomas by in situ hybridization and reverse transcription-PCR. PRL adenomas had a significantly higher density of labeling compared to other adenoma types. Although there was no difference in the levels of PRL-R mRNA in PRL adenomas from men and premenopausal and postmenopausal women, patients treated with bromocriptine before pituitary surgery had significantly lower levels of PRL-R compared to all other groups. These results indicate that in the normal pituitary, PRL and LH cells have the highest level of PRL-R mRNA, whereas PRL adenomas have significantly higher levels of PRL-R mRNA than other types of adenomas, and bromocriptine treatment decreases the levels of PRL-R mRNA in PRL adenomas.

scholarly journals Somatic coliphages and bacterial indicators of bathing water quality in the beaches of Gipuzkoa, Spain

2007 ◽  
Vol 5 (3) ◽  
pp. 417-426 ◽  
Author(s):  
Jesús M. Ibarluzea ◽  
Belén Moreno ◽  
Elena Serrano ◽  
Karmele Larburu ◽  
María J. Maiztegi ◽  
...  
Keyword(s):  
Water Quality ◽  
Basque Country ◽  
Water Quality Monitoring ◽  
Bacterial Indicators ◽  
Somatic Coliphages ◽  
E Coli ◽  
Bathing Water ◽  
Bathing Waters ◽  
Viral Indicators ◽  
Bathing Water Quality

Monitoring the quality of the bathing waters of Gipuzkoa (the Basque Country, Spain) makes it possible to assess the suitability of its 15 beaches for bathing throughout each season. In 1998, the parameters E. coli, somatic coliphages (SOMCPH) and F-specific RNA bacteriophages (FRNAPH) were incorporated into the bathing water quality monitoring system. This enabled the study of the link between bacterial and viral indicators as well as the analysis of the ratios between both types of indicators in waters with different levels of pollution. Although bacterial indicators (total coliforms (TC) and faecal coliforms (FC)) and enterococci showed a strong correlation between them, the correlations between the viral indicators and between the viral and bacterial indicators were weaker, though significant in all cases. The ratio between SOMCPH and E. coli indicates that at low levels of bacterial pollution (E coli &lt;100 MPN/100 ml) SOMCPH outnumber E coli. In contrast, at higher levels of pollution (E coli &gt;100 MPN/100 ml), SOMCPH numbers are lower than those of E-coli. The data reveal the presence of viral indicators in waters classified as suitable for bathing by the European Directive and alert us to their suitability.

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