scholarly journals Building a molecular typing protocol for rs1801133 based on real-time PCR HRM technique

2019 ◽  
Vol 2 (3) ◽  
pp. 5-13
Author(s):  
Linh Thi Nhut Tran ◽  
My Thi Huynh Nguyen ◽  
Linh Nguy Hoang Le ◽  
Khoa Dang Le ◽  
Minh Hoang Nhat Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Neurological Diseases ◽  
Human Diseases ◽  
Chromosome 1 ◽  
Nucleotide Sequencing ◽  
Sequencing Method ◽  
Mgcl2 Concentration ◽  
Clear Differentiation ◽  
The Relationship

rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the realtime PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.

scholarly journals Decreased miRNA-146A in Glioblastoma Multiforme and Regulation of Cell Proliferation and Apoptosis by Target Notch1

2016 ◽  
Vol 31 (3) ◽  
pp. 270-275 ◽  
Author(s):  
Hong-qi Hu ◽  
Lai-guang Sun ◽  
Wu-jun Guo
Keyword(s):  
Glioblastoma Multiforme ◽  
Cell Viability ◽  
Real Time ◽  
Real Time Pcr ◽  
Glioma Cells ◽  
Viability Analysis ◽  
Nontumor Tissue ◽  
Proliferation And Apoptosis ◽  
A Cell ◽  
The Relationship

Objective The primary purpose of this paper is to investigate the relationship between the microRNA 146a (miR-146a) and the proliferation of cells occurring in glioblastoma multiforme. The secondary purpose of the paper is to investigate abnormalities of expression in miR-146a. Methods A real-time PCR assay was used to investigate the abnormal expression of miR-146a in glioma and adjacent tissue. Lipofection was used to transfect a mimic of miR-146a and induce the upregulation of miR-146a. Real-time PCR was used to observe the expression level of miR-146a. A cell viability analysis was conducted using MTT. A luciferase report vector was used to identify potential targets for miR-146a. Results The miR-146a component was found to be downregulated in glioma tissue compared with adjacent nontumor tissue (p<0.05). The upregulation of miR-146a in glioma cells through miR-146a mimic transfection led to reduction of cell viability and to an increase in the percentage of apoptosis. Notch1 was the name of the potential targeted gene for miR-146a in glioma. Conclusions The study found that the presence of miR-146a potentially affected the proliferation of glioma cells by regulating the rate of Notch1 expression.

Variance Calculations for Quantitative Real-Time PCR Experiments with Multiple Levels of Replication

2013 ◽  
Vol 825 ◽  
pp. 172-176
Author(s):  
Susana Soto-Rojo ◽  
Gary Glonek ◽  
Cecilia Demergasso ◽  
Pedro A. Galleguillos ◽  
Patty Solomon ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Industrial Process ◽  
Error Variance ◽  
Low Grade ◽  
Sulphide Ores ◽  
Polymerase Chain ◽  
Frequent Sampling ◽  
The Relationship ◽  
Proportional Reduction

Heap bioleaching is an established technology for recovering copper from low-grade sulphide ores. Recently, genetics-based approaches have been employed to characterize mineral-processing bacteria. In these approaches, data analysis is a key issue. Consequently, it is of fundamental importance to provide adequate mathematical models and statistical tools to draw reliable conclusions. The present work relates to current studies of the consortium of organisms inhabiting the bioleaching heap of the Escondida mine in Northern Chile. These studies aim to describe and understand the relationship between the dynamics of the community and the performance of the industrial process. Here, we consider a series of quantitative real-time polymerase chain reaction (PCR) experiments performed to quantify six different microorganisms at various stages of the bioleaching cycle. Establishing the reliability of the data obtained by real-time PCR requires the estimation of the error variance at several different levels. The results obtained show that the sampling component of the error variance is the dominant source of variability for most microorganisms. An estimate for the proportional reduction in residual standard deviation from the use of extraction and real-time PCR triplicates was found to range from 3% to 27% for the different organisms. This result suggests that triplicate assays would produce only a modest reduction in error variance compared to more frequent sampling from the heap.

scholarly journals Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead Legionella in Biofilm Samples

2014 ◽  
Vol 7 ◽  
pp. MBI.S17723 ◽  
Author(s):  
Michael J. Taylor ◽  
Richard H. Bentham ◽  
Kirstin E. Ross
Keyword(s):  
Public Health ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Propidium Monoazide ◽  
Factors Affecting ◽  
Conventional Culture ◽  
Target Dna ◽  
The Relationship ◽  
Conventional Culture Method

Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes.

scholarly journals Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests

2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Anh Duc Truong ◽  
Duc Viet Ly ◽  
Thi Hao Vu ◽  
Van Tuan Hoang ◽  
Thi Chinh Nguyen ◽  
...  
Keyword(s):  
Binding Site ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
False Negative ◽  
African Swine Fever ◽  
Molecular Diagnostic ◽  
Conventional Pcr ◽  
Sequencing Method ◽  
Single Mismatch

Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result.Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection.Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells.Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study.Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences,  particularly for epidemiological surveillance of ASF. Keywords: African swine fever, PCR, Pigs, Real-time PCR, Vietnam

scholarly journals The multidrug resistance can be reversed for the decrease of P-gp and LRP by inhibiting PI3K/Akt/NF-κB signal pathway in nasopharynx carcinoma

2020 ◽  
Vol 40 (5) ◽  
Author(s):  
Jin Liu ◽  
Mingyi Zhu ◽  
Yun Feng ◽  
Qianli Tang ◽  
Meng Xu
Keyword(s):  
Multidrug Resistance ◽  
Nasopharyngeal Carcinoma ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Carcinoma Cell ◽  
Signal Pathway ◽  
Nasopharyngeal Carcinoma Cell ◽  
Cellular Signal ◽  
The Relationship

Abstract Aim: To investigate the relationship between PI3K/Akt/NF-κB cellular signal pathway and the expression of P-gp and LRP in multidrug resistance (MDR) cell of nasopharyngeal carcinoma. Method: The PI3K, p-Akt and NF-κB/p65 as the activity of PI3K/Akt/NF-κB were detected by Western blot. The expressions of LRP and P-gp were detected by Western blot and real-time PCR. Result: The RIs of CNE/DDP group to DDP, 5-Fu, VCR, ADR and PTX were 35.04, 18.14, 24.13, 12.00 and 10.18, respectively. The RIs of LY-294002 group were 11.77, 5.83, 3.07, 3.86 and 3.34, and PDTC group were 11.08, 6.55, 7.66, 2.18 and 4.05. The expressions of PI3K, p-Akt and NF-κBp65, LRP and P-gp were increased and mRNA of LRP and P-gp were up-regulated in CNE/DDP. The expression of p-Akt in LY-294002 group was down-regulated. The expression of NF-κB p65 in PDTC group was decreased. The mRNA of LRP and P-gp in LY-294002 group and PDTC group were decreased. Conclusion: MDR of nasopharyngeal carcinoma cell can be regulated by activating PI3K/Akt/NF-κB signal pathway and then increase the expression of P-gp and LRP. The MDR of nasopharyngeal carcinoma cell can be reversed by inhibiting PI3K/Akt/NF-κB signal pathway.

scholarly journals Quantification and conceptual validation of the inoculum potential of Sclerotinia sclerotiorum in soybean and bean seeds

2021 ◽  
Vol 43 ◽  
Author(s):  
Sueny Kelly Santos de França ◽  
Carolina da Silva Siqueira ◽  
Marina de Resende Faria Guimarães ◽  
José da Cruz Machado
Keyword(s):  
Seed Germination ◽  
Real Time ◽  
Molecular Analysis ◽  
Sclerotinia Sclerotiorum ◽  
Real Time Pcr ◽  
White Mold ◽  
Inoculum Potential ◽  
Severe Damage ◽  
The World ◽  
The Relationship

Abstract: The fungus Sclerotinia sclerotiorum, the causal agent of white mold, is widespread throughout the world. The disease is considered to be one of the major diseases of soybean and bean crops in Brazil. The pathogen S. sclerotiorum is spread by soybean and bean seeds both in the form of sclerotia and dormant mycelium inside the seeds. The objective of this work was to evaluate the relationship between different potentials of S. sclerotiorum in soybean and bean seeds and the performance of these seeds, as well as to verify the localization and quantification of the inoculum of the pathogen in the seeds inoculated by Real-time PCR (qPCR), validating the term inoculum potential. Soybean and bean seeds were inoculated with the fungus by the osmotic conditioning method based on the exposure of the seeds to the fungus for periods of 24 h, 48 h, 72 h, and 96 h. Molecular analysis was carried out by means of qPCR in whole seeds and dissected in the integument, cotyledon and embryonic axis. The results showed that the effects of S. sclerotiorum on seed germination and vigor were progressive and proportional to the increases in inoculum potentials, since there was more severe damage to the seeds and consequently to the emerged plants at the highest potential (P96). The inoculum of the pathogen was found in all parts of the evaluated seeds, even at its lowest inoculum potential (P24), with an increasing DNA concentration, and the integument obtained a greater amount of DNA than the embryo, in comparison.

Quantitative real-time PCR for rhinovirus, and its use in determining the relationship between TCID50 and the number of viral particles

2011 ◽  
Vol 171 (1) ◽  
pp. 212-218 ◽  
Author(s):  
Lorne A. Sachs ◽  
David Schnurr ◽  
Shigeo Yagi ◽  
Marrah E. Lachowicz-Scroggins ◽  
Jonathan H. Widdicombe
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Viral Particles ◽  
The Relationship

scholarly journals Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses

2016 ◽  
Vol 29 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Amaresh Das ◽  
Gordon Ward ◽  
Andre Lowe ◽  
Lizhe Xu ◽  
Karen Moran ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Positive Control ◽  
Nucleotide Sequencing ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Highly Sensitive ◽  
Development And Validation

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91–99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28–1.06 and 0.01–0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.

scholarly journals Zishen Qingre Tongluo Formula Improves Renal Fatty Acid Oxidation and Alleviated Fibrosis via the Regulation of the TGF-β1/Smad3 Signaling Pathway in Hyperuricemic Nephrology Rats

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Peng Liu ◽  
Chen Wang ◽  
Yun Wang ◽  
Honghong Zhang ◽  
Baoli Liu ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Real Time ◽  
Signaling Pathway ◽  
Fatty Acid Oxidation ◽  
Western Blotting ◽  
Real Time Pcr ◽  
Acid Oxidation ◽  
Immunohistochemistry Staining ◽  
The Relationship ◽  
Tgf Β1

Hyperuricemia, an independent risk factor for ensuing chronic kidney disease (CKD), contributed to tubulointerstitial fibrosis and insufficiency of renal fatty acid oxidation. Many studies have shown that renal fatty acid oxidation dysfunction is related to the TGF-β1/Smad3 signaling pathway. We experimented the effects of Zishen Qingre Tongluo Formula (ZQTF) on the adenine/yeast-induced HN rats and uric acid-induced renal mouse tubular epithelial cells (mTECs), determined whether this effect was related to the TGF-β1/Smad3 signaling pathway, and further investigated the relationship between this effect and renal fatty acid oxidation. Rats were given intraperitoneally with adenine (100 mg/kg) and feed chow with 10% yeast for 18 days and then received ZQTF (12.04 g/kg/day) via intragastric gavage for eight weeks. The TGF-β1/Smad3 signaling pathway and renal fatty acid oxidation protein were detected by using western blotting, real-time PCR, and immunohistochemistry staining. mTECs induced by UA were used to investigate the relationship between the TGF-β1/Smad3 signaling pathway and renal fatty acid oxidation. After treatment with ZQTF, levels of UA, 24 h UTP, BUN, and Scr were significantly decreased and histologic injuries were visibly ameliorated in HN rats. Western blotting, real-time PCR, and immunohistochemistry staining revealed that PGC-1α, PPARγ, and PPARα significantly increased, and fibronectin, collagen 1, and P-Smad3 significantly decreased in HN rats and UA-induced mTECs after ZQTF treatment. SIS3 (a specific inhibitor of Smad3) treatment significantly increased the expressions of PGC-1α, PPARγ, and PPARα and decreased the expressions of fibronectin, collagen 1, and P-Smad3 in UA-induced mTECs. Our study demonstrated that ZQTF exhibited renoprotective effects by promoting renal fatty acid oxidation via the regulation of the TGF-β1/Smad3 signaling pathway. Thus, the present results indicated that ZQTF was a novel antifibrotic strategy for hyperuricemic nephropathy.

Sign in / Sign up

Export Citation Format

Share Document