scholarly journals Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of novel-goose parvovirus in vivo

2021 ◽  
Author(s):  
Haibin Ma ◽  
Yahui Li ◽  
Junzheng Yang
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Bursa Of Fabricius ◽  
Pcr Assay ◽  
Standard Curve ◽  
Goose Parvovirus ◽  
Detection And Quantification

Objectives: To develop a sensitive, highly specific fluorescent quantitative real-time PCR assay for accurate detection and quantification of novel-goose parvovirus (N-GPV) in vitro and in vivo. Methods: Specific primers was designed based on N-GPV inverted terminal repeats region; virus RNA (DFV, NDV, AIV, DHV-1, DHV-3) and virus DNA (MDPV, GPV, N-GPV) were extracted, cDNA (DFV, NDV, AIV, DHV-1, DHV-3) were prepared from viral RNAs using M-MLV Reverse Transcriptase, and prepared cDNA (DFV, NDV, AIV, DHV-1, DHV-3) and DNA (MDPV, GPV, N-GPV) amplified by real-time PCR; the sensitivity, specificity and reproducibility of established real-time PCR methods were evaluated, and finally we validated the reliability of real-time PCR methods in ducklings models in vivo. Results: The standard curve of established real-time PCR had a good linearity (slope was -0.3098, Y-intercept was 37.865, efficiency of standard curve was 0.995); the detection limit of established real-time PCR for N-GPV was 10 copies/reaction. The sensitivity of real-time PCR was 10 copies/uL, which was 1000 times higher than conventional gel-based PCR assay. The results of intra-assay CVs (0.04-0.74%) and inter-assay CVs (0.16-0.53%) showed that the real-time PCR assay had an excellent repeatability. This method also could efficiently detect viral load in heart, liver, spleen, lung, kidney, pancreas, bursa of Fabricius, brain, blood and excrement from ducklings models after N-GPV infection from 6h to 28 days, which could provided us a dynamic distribution observation of N-GPV viral load using this real-time PCR assay in vivo. Conclusion: In the study, we developed a high sensitive, specific and reproducible real-time PCR assay for N-GPV detection. The established real-time PCR assay was suitable for parvovirus detection and quantification simultaneously, no matter sample obtained from blood, internal organs or ileac contents; the present work may provide insight into the pathogenesis of N-GPV and will contributes to better understanding of this newly emerged novel GPV related virus in cherry valley ducks.

scholarly journals A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae

2020 ◽  
Vol 119 (11) ◽  
pp. 3909-3913
Author(s):  
Zaida Rentería-Solís ◽  
Tran Nguyen-Ho-Bao ◽  
Shahinaz Taha ◽  
Arwid Daugschies
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Small Subunit ◽  
Sybr Green ◽  
Pcr Assay ◽  
Standard Curve ◽  
Trichomonas Gallinae ◽  
Host Interaction

Abstract Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

Development of Primer-Probe Energy Transfer real-time PCR for the detection and quantification of porcine circovirus type 2

2009 ◽  
Vol 57 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Ádám Bálint ◽  
Miklós Tenk ◽  
Zoltán Deim ◽  
Thomas Rasmussen ◽  
Åse Uttenthal ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Pcr Assay ◽  
Detection And Quantification

A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in post-weaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 107copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

scholarly journals One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

2018 ◽  
Vol 22 (5) ◽  
pp. 418-423
Author(s):  
Elisabete Andrade ◽  
Daniele Rocha ◽  
Marcela Fontana-Maurell ◽  
Elaine Costa ◽  
Marisa Ribeiro ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
One Step ◽  
Detection And Quantification

Development of a standardized real time PCR for Torque teno viruses (TTV) viral load detection and quantification: A new tool for immune monitoring

2018 ◽  
Vol 105 ◽  
pp. 118-127 ◽  
Author(s):  
Dorian Kulifaj ◽  
Bénédicte Durgueil-Lariviere ◽  
Faustine Meynier ◽  
Eliza Munteanu ◽  
Nicolas Pichon ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Immune Monitoring ◽  
Load Detection ◽  
Detection And Quantification

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/β-catenin

2020 ◽  
Vol 48 (02) ◽  
pp. 341-356
Author(s):  
Chiu-Mei Lin ◽  
Wei-Jen Fang ◽  
Bao-Wei Wang ◽  
Chun-Ming Pan ◽  
Su-Kiat Chua ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Real Time ◽  
Real Time Pcr ◽  
Myocardial Infarct ◽  
Epigallocatechin Gallate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cardiomyocytes

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/[Formula: see text]-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and [Formula: see text]-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50[Formula: see text]mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and [Formula: see text]-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/[Formula: see text]-catenin pathways.

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