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

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.

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

10.1101/2021.07.09.451858 ◽

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.

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Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A

Journal of Virological Methods ◽

10.1016/j.jviromet.2007.03.019 ◽

2007 ◽

Vol 144 (1-2) ◽

pp. 57-64 ◽

Cited By ~ 40

Author(s):

Wee Theng Ong ◽

Abdul Rahman Omar ◽

Aini Ideris ◽

Sharifah Syed Hassan

Keyword(s):

Influenza Virus ◽

Real Time ◽

Virus Type ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Type A ◽

Sybr Green ◽

Pcr Assay ◽

H9n2 Influenza Virus

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

Frontiers in Oncology ◽

10.3389/fonc.2021.752573 ◽

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.

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

Protocols used in Molecular Biology ◽

10.2174/9789811439315120010006 ◽

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.

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

The American Journal of Chinese Medicine ◽

10.1142/s0192415x20500172 ◽

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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Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

BioMed Research International ◽

10.1155/2019/5715180 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Cyril C. Y. Yip ◽

Siddharth Sridhar ◽

Kit-Hang Leung ◽

Andrew K. W. Cheng ◽

Kwok-Hung Chan ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Test Performance ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Clinical Specimens ◽

Varicella Zoster ◽

Simplex Virus ◽

Hsv 1

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

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A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

Journal of Clinical Microbiology ◽

10.1128/jcm.01986-20 ◽

2020 ◽

Vol 59 (1) ◽

pp. e01986-20

Author(s):

Ibne Karim M. Ali ◽

Shantanu Roy

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Small Subunit ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Rdna Sequences ◽

Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

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