Development of In situ hybridization and real-time PCR assays for the detection of Hepatospora eriocheir , a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

2016 ◽  
Vol 40 (7) ◽  
pp. 919-927 ◽  
Author(s):  
Z F Ding ◽  
J Q Chen ◽  
J Lin ◽  
X S Zhu ◽  
G H Xu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr ◽  
Eriocheir Sinensis ◽  
Chinese Mitten Crab ◽  
Mitten Crab ◽  
Pcr Assays

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

2005 ◽  
Vol 68 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Han Vervaeren ◽  
Kurt De Wilde ◽  
Jorg Matthys ◽  
Nico Boon ◽  
Lutgarde Raskin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr

scholarly journals OCT4B1 Regulates the Cellular Stress Response of Human Dental Pulp Cells with Inflammation

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Lu Liu ◽  
Rong Huang ◽  
Ruiqi Yang ◽  
Xi Wei
Keyword(s):  
In Situ Hybridization ◽  
Survival Rate ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Dental Pulp ◽  
Immunofluorescence Staining ◽  
Dental Pulp Cells ◽  
Pulp Cells

Introduction. Infection and apoptosis are combined triggers for inflammation in dental tissues. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel spliced variant of OCT4 family, could respond to the cellular stress and possess antiapoptotic property. However, its specific role in dental pulpitis remains unknown. Methods. To investigate the effect of OCT4B1 on inflammation of dental pulp cells (DPCs), its expression in inflamed dental pulp tissues and DPCs was examined by in situ hybridization, real-time PCR, and FISH assay. OCT4B1 overexpressed DPCs model was established, confirmed by western blot and immunofluorescence staining, and then stimulated with Lipopolysaccharide (LPS). Apoptotic rate was determined by Hoechst/PI staining and FACS. Cell survival rate was calculated by CCK8 assay. Results. In situ hybridization, real-time PCR, and FISH assay revealed that OCT4B1 was extensively expressed in inflamed dental pulp tissues and DPCs with LPS stimulation. Western blot and immunofluorescence staining showed the expression of OCT4B1 and OCT4B increased after OCT4B1 transfection. Hoechst/PI staining and FACS demonstrated that less red/blue fluorescence was detected and apoptotic percentage decreased (3.45%) after transfection. CCK8 demonstrated that the survival rate of pCDH-OCT4B1-flag cells increased. Conclusions. OCT4B1 plays an essential role in inflammation and apoptosis of DPCs. OCT4B might operate synergistically with OCT4B1 to reduce apoptosis.

scholarly journals Histological Lesions and Replication Sites of PCV3 in Naturally Infected Pigs

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1520
Author(s):  
Elisa Rigo De Conti ◽  
Talita Pilar Resende ◽  
Lacey Marshall-Lund ◽  
Albert Rovira ◽  
Fabio Augusto Vannucci
Keyword(s):  
In Situ Hybridization ◽  
Lymph Nodes ◽  
Real Time ◽  
Real Time Pcr ◽  
Systemic Vasculitis ◽  
Tissue Type ◽  
Lymphoid Tissues ◽  
Associated Diseases ◽  
Spleen And Lymph Nodes

Porcine circovirus type 3 (PCV3) has been recently described as a potential cause of abortions and systemic vasculitis in pigs. Although the virus has been detected by real-time PCR in several porcine tissues from countries worldwide, PCV3-associated diseases have not been satisfactorily clarified. The objective of this study was to investigate the association between the presence of PCV3 mRNA detected by in situ hybridization (ISH) within histological lesions and PCV3 DNA detected by real-time PCR in naturally infected pigs. A total of 25 PCV3 PCR-positive cases were analyzed. Formalin-fixed tissues from these cases were evaluated for histologic lesions and for ISH-RNA positive signals for PCV3. The most frequent tissue type with histopathologic lesions was heart, 76.2%, with lymphoplasmacytic myocarditis and epicarditis as the most frequent lesions observed. Lymphoplasmacytic interstitial pneumonia was also a frequent finding, 47.6%. There were also lesions in kidney, liver, spleen and lymph nodes. PCV3-ISH-RNA positive signals were mostly observed in association with lymphoplasmacytic inflammatory infiltrate in various tissues, including arteries. Based on our results, the minimum set of specimens to be submitted for histopathology and mRNA in situ hybridization to confirm or exclude a diagnosis of PCV3 are heart, lung and lymphoid tissues (i.e., spleen and lymph nodes), especially for differential diagnosis related with PCV2-associated diseases.

scholarly journals New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4165-4169 ◽  
Author(s):  
Miguel Gueimonde ◽  
Satu Tölkkö ◽  
Teemu Korpimäki ◽  
Seppo Salminen
Keyword(s):  
In Situ Hybridization ◽  
Detection Limit ◽  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Fluorescent In Situ Hybridization ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Lanthanide Chelate

ABSTRACT The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 104 cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.

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