Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

2007 ◽  
Vol 363 (1) ◽  
pp. 171-177 ◽  
Author(s):  
Ricky R. Lareu ◽  
Karthik S. Harve ◽  
Michael Raghunath
Keyword(s):  
Rt Pcr ◽  
Intracellular Environment

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

scholarly journals Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 66
Author(s):  
Zoltán László ◽  
Péter Pankovics ◽  
Gábor Reuter ◽  
Attila Cságola ◽  
Ádám Bálint ◽  
...  
Keyword(s):  
Novel Species ◽  
Phylogenetic Analyses ◽  
Interspecies Transmission ◽  
Background Information ◽  
Type Ii ◽  
Rt Pcr ◽  
Faecal Samples ◽  
The Family ◽  
Domestic Livestock

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain “neglected” genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species “Bopivirus B” in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine–caprine interspecies transmission of certain bopiviruses.

scholarly journals Overexpression of P-glycoprotein, MRP2, and CYP3A4 impairs intestinal absorption of octreotide in rats with portal hypertension

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaoyu Sun ◽  
Shunxiong Tang ◽  
Binbin Hou ◽  
Zhijun Duan ◽  
Zhen Liu ◽  
...  
Keyword(s):  
Liver Cirrhosis ◽  
Portal Hypertension ◽  
Western Blot ◽  
In Vitro Experiments ◽  
Rt Pcr ◽  
P Glycoprotein ◽  
Intestinal Microsomes ◽  
First Pass

Abstract Background Portal hypertension (PH) is the main cause of complications and death in liver cirrhosis. The effect of oral administration of octreotide (OCT), a drug that reduces PH by the constriction of mesenteric arteries, is limited by a remarkable intestinal first-pass elimination. Methods The bile duct ligation (BDL) was used in rats to induce liver cirrhosis with PH to examine the kinetics and molecular factors such as P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and cytochrome P450 3A4 (CYP3A4) influencing the intestinal OCT absorption via in situ and in vitro experiments on jejunal segments, transportation experiments on Caco-2 cells and experiments using intestinal microsomes and recombinant human CYP3A4. Moreover, RT-PCR, western blot, and immunohistochemistry were performed. Results Both in situ and in vitro experiments in jejunal segments showed that intestinal OCT absorption in both control and PH rats was largely controlled by P-gp and, to a lesser extent, by MRP2. OCT transport mediated by P-gp and MRP2 was demonstrated on Caco-2 cells. The results of RT-PCR, western blot, and immunohistochemistry suggested that impaired OCT absorption in PH was in part due to the jejunal upregulation of these two transporters. The use of intestinal microsomes and recombinant human CYP3A4 revealed that CYP3A4 metabolized OCT, and its upregulation in PH likely contributed to impaired drug absorption. Conclusions Inhibition of P-gp, MRP2, and CYP3A4 might represent a valid option for decreasing intestinal first-pass effects on orally administered OCT, thereby increasing its bioavailability to alleviate PH in patients with cirrhosis.

scholarly journals Interaction of periodontitis and orthodontic tooth movement—an in vitro and in vivo study

2021 ◽  
Author(s):  
Birgit Rath-Deschner ◽  
Andressa V. B. Nogueira ◽  
Svenja Beisel-Memmert ◽  
Marjan Nokhbehsaim ◽  
Sigrun Eick ◽  
...  
Keyword(s):  
Alveolar Bone ◽  
Tooth Movement ◽  
Mechanical Strain ◽  
Orthodontic Tooth Movement ◽  
Fusobacterium Nucleatum ◽  
In Vivo Study ◽  
Rt Pcr ◽  
Periodontal Inflammation

Abstract Objectives The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). Materials and methods The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. Results Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. Conclusions Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. Clinical relevance Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

scholarly journals Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II

2015 ◽  
Vol 45 (4) ◽  
pp. 704-710 ◽  
Author(s):  
Melânia Lazzari Rigo ◽  
Andressa Minussi Pereira Dau ◽  
Werner Giehl Glanzner ◽  
Manoel Martins ◽  
Renato Zanella ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Media ◽  
Mrna Levels ◽  
Ang Ii ◽  
Rt Pcr ◽  
Steroidogenic Enzymes ◽  
Theca Cells ◽  
Mrna Profile ◽  
Dose Response Effect

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells

Effect of sub-inhibitory concentrations of cefepime on biofilm formation by Pseudomonas aeruginosa

2021 ◽  
pp. 1-8
Author(s):  
Soheir A.A. Hagras ◽  
Alaa El-Dien M.S. Hosny ◽  
Omneya M. Helmy ◽  
Mounir M. Salem-Bekhit ◽  
Faiyaz Shakeel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Clinical Isolates ◽  
Polymeric Chain ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Encoding Gene ◽  
Fimbrial Protein

This study investigated the effect of cefepime at sub-minimum inhibitory concentrations (sub-MICs) on in vitro biofilm formation (BF) by clinical isolates of Pseudomonas aeruginosa. The effect of cefepime at sub-MIC levels (½–1/256 MIC) on in vitro BF by six clinical isolates of P. aeruginosa was phenotypically assessed following 24 and 48 h of challenge using the tissue culture plate (TCP) assay. Quantitative real-time polymeric chain reaction (qRT-PCR) was employed to observe the change in expression of three biofilm-related genes, namely, a protease-encoding gene (lasA), fimbrial protein-encoding gene (cupA1), and alginate-encoding gene (algC), in a weak biofilm-producing strain of P. aeruginosa following 24 and 48 h of challenge with sub-MICs of cefepime. The BF morphology in response to cefepime was imaged using scanning electron microscopy (SEM). The TCP assay showed strain-, time-, and concentration-dependent changes in in vitro BF in P. aeruginosa following challenge with sub-MICs of cefepime, with a profound increase in strains with inherently no or weak biofilm-producing ability. RT-PCR revealed time-dependent upregulation in the expression of the investigated genes following challenge with ½ and ¼ MIC levels, as confirmed by SEM. Cefepime at sub-MICs could upregulate the expression of BF-related genes and enhance BF by P. aeruginosa clinical isolates.

Verificación de conformidad de dispositivos médicos de diagnóstico in vitro manufacturados en Chile para la detección del material genético de SARS-CoV-2 desde muestras de hisopado

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Daniel Escobar ◽  
Ricardo Manzo ◽  
Rodrigo Puentes ◽  
Diego Diaz-Dinamarca ◽  
Gonzalo Marambio ◽  
...  
Keyword(s):  
Rt Pcr

La pandemia por coronavirus 2 del síndrome respiratorio agudo severo (SARS-CoV-2), que causa la enfermedad de coronavirus 2019 (COVID-19), trajo consigo grandes exigencias a nivel científico; una de ellas es la oportuna detección de pacientes contagiados. En este contexto, los dispositivos médicos de diagnóstico in vitro (IVD, en sus siglas en inglés) han constituido una alternativa esencial en el manejo y control de la pandemia en todo el mundo, por su sensibilidad, especificidad, y alto rendimiento en el número de muestras analizadas. Gran parte de estos kits de uso diagnóstico fueron autorizados para uso de emergencia por agencias regulatorias, basando esta autorización en la revisión documental de los informes de validación de cada dispositivo. En ese sentido, la realización de una verificación de conformidad constituye una importante herramienta para evaluar los dispositivos médicos IVD que están siendo utilizados para la detección de pacientes con COVID-19. En este estudio se presentan los resultados de verificación de conformidad realizadas en el Instituto de Salud Pública de Chile (ISP) de 5 dispositivos médicos IVD con fundamento genético y de fabricación nacional: 3 basados en la técnica de reacción en cadena de la polimerasa con transcripción reversa (RT-PCR, en sus siglas en inglés) en tiempo real y 2 basados en la técnica de amplificación isotérmica mediada por bucle con transcripción reversa (RT-LAMP, en sus siglas en inglés). De los dispositivos médicos IVD evaluados, un 80% (4/5) fueron verificados en conformidad y un 20% (1/5) fue rechazado por no cumplir con los criterios de aceptabilidad en términos de sensibilidad y especificidad. Estos resultados preliminares demuestran que el protocolo de verificación de conformidad implementado en el ISP permite identificar los dispositivos médicos IVD que cumplen con lo declarado por el fabricante de aquellos que no cumplen y terminan siendo rechazados.

scholarly journals Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR

1997 ◽  
Vol 82 (6) ◽  
pp. 1926-1931 ◽  
Author(s):  
Nobuharu Fujii ◽  
Takeshi Shibata ◽  
Sachiko Homma ◽  
Haruo Ikegami ◽  
Kazuo Murakami ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Human Lymphocytes ◽  
Mrna Level ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Receptor Mrna ◽  
Exercise Induced ◽  
Induced Changes ◽  
Receptor Mrna Level

Fujii, Nobuharu, Takeshi Shibata, Sachiko Homma, Haruo Ikegami, Kazuo Murakami, and Hitoshi Miyazaki. Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR. J. Appl. Physiol. 82(6): 1926–1931, 1997.—Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to clarify whether dynamic exercise-induced increases in β-adrenergic-receptor (β-AR) number in human lymphocytes are accompanied by increases in the β-AR mRNA level. Sixteen healthy subjects performed cycle ergometry until exhaustion. Before and immediately after exercise, peripheral blood was drawn from a forearm vein for preparation of lymphocytes. Both the β-AR mRNA level and the β-AR number were significantly increased by exercise. The changes in β-AR mRNA level and β-AR number were significantly correlated ( r = 0.63, P < 0.01). This finding suggests that a rapid increase in β-AR mRNA level might be an early adaptive response of the sympathetic nervous system to dynamic exercise. In vitro incubation of lymphocytes with epinephrine had no effect on β-AR mRNA levels, nor did adenosine 3′,5′-cyclic monophosphate, protein kinase C, or intracellular Ca2+ increase the β-AR mRNA level in vitro. Therefore, it appears that other mechanisms underlie the exercise-induced elevation of β-AR mRNA levels in human lymphocytes.

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