scholarly journals Establishment and Application of Ligation Reaction-Based Method for Quantifying MicroR-156b

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuxuan He ◽  
Likun Long ◽  
Wei Yan ◽  
Liming Dong ◽  
Wei Xia ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Dna Probes ◽  
Universal Primers ◽  
Quantitative Detection ◽  
Specific Method ◽  
Biological Regulation ◽  
Ligation Reaction ◽  
Complex Design ◽  
Modified Dna ◽  
Regulation Mechanisms

Microribonucleic acids (miRNAs) play significant roles in the regulation of biological processes and in responses to biotic or abiotic environmental stresses. Therefore, it is necessary to quantitatively detect miRNAs to understand these complicated biological regulation mechanisms. This study established an ultrasensitive and highly specific method for the quantitative detection of miRNAs using simple operations on the ground of the ligation reaction of ribonucleotide-modified deoxyribonucleic acid (DNA) probes. This method avoids the complex design of conventional reverse transcription. In the developed assay, the target miRNA miR156b was able to directly hybridize the two ribonucleotide-modified DNA probes, and amplification with universal primers was achieved following the ligation reaction. As a result, the target miRNA could be sensitively measured even at a detection limit as low as 0.0001 amol, and differences of only a single base could be detected between miR156 family members. Moreover, the proposed quantitative method demonstrated satisfactory results for overexpression-based genetically modified (GM) soybean. Ligation-based quantitative polymerase chain reaction (PCR) therefore has potential in investigating the biological functions of miRNAs, as well as in supervising activities regarding GM products or organisms.

scholarly journals Real-Time PCR for Quantitative Detection of Chamois (Rupicapra rupicapra) and Pyrenean Ibex (Capra pyrenaica) in MeatMixtures

2008 ◽  
Vol 91 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Violeta Fajardo ◽  
Isabel Gonzlez ◽  
Irene Martn ◽  
Mara Rojas ◽  
Pablo E Hernndez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Quantitative Polymerase Chain Reaction ◽  
Universal Primers ◽  
Quantitative Detection ◽  
12S Rrna ◽  
Rrna Gene ◽  
Capra Pyrenaica ◽  
Loop Region

Abstract A real-time quantitative polymerase chain reaction (PCR) technique was developed for the quantification of chamois and pyrenean ibex DNAs in meat mixtures by using a SYBR green detection platform. Two species-specific systems and a eukaryotic endogenous system were combined in the real-time PCR approach to quantify the target species. In the specific systems, a 133 base pair (bp) fragment of the 12S rRNA gene was amplified from chamois DNA, and an 88 bp fragment from the D-loop region was amplified from pyrenean ibex DNA. In the endogenous system, universal primers amplified a 141 bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The threshold cycle values obtained with the 18S rRNA primers were used to normalize those obtained from chamois- or pyrenean ibex-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat-treated binary mixtures of chamois and pyrenean ibex meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target DNAs in the range of 0.10.8, depending on the species and treatment of the meat samples.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals Effects of Contemporary Irrigant Activation Schemes and Subsequent Placement of an Interim Dressing on Bacterial Presence and Activity in Root Canals Associated with Asymptomatic Apical Periodontitis

2020 ◽  
Vol 9 (3) ◽  
pp. 854 ◽  
Author(s):  
Alexandre P. L. Carvalho ◽  
Laura C. L. Nardello ◽  
Fernanda S. Fernandes ◽  
Fernanda P. Bruno ◽  
Luiza R. Paz ◽  
...  
Keyword(s):  
Calcium Hydroxide ◽  
Root Canal ◽  
Quantitative Polymerase Chain Reaction ◽  
Apical Periodontitis ◽  
Universal Primers ◽  
Rrna Gene ◽  
Root Canal Preparation ◽  
Root Canals ◽  
Mechanical Preparation ◽  
Root Canal Disinfection

New tools for activating endodontic irrigants have evolved, yet their impact on root canal disinfection, in comparison to the passive placing of an inter-visit medication, have not yet been fully elucidated. The use of DNA- and rRNA-based methods may cast some new light on this issue, as they allow a comparison to be made between microbial presence and activity. Therefore, the aim of this single-arm intervention trial is to evaluate the antibacterial effect of endodontic procedures using both molecular methods. Root canal samples were obtained from 20 patients with asymptomatic apical periodontitis after each treatment step: access cavity, chemo-mechanical preparation, adjunctive procedures (XP-endo Finisher file and passive ultrasonic irrigation), calcium hydroxide medication, and 2nd-visit root canal preparation. DNA and cDNA from the samples were subjected to quantitative polymerase chain reaction with universal primers for the bacterial 16S rRNA gene. Chemo-mechanical preparation promoted a drastic reduction in bacterial levels and activity, whereas the adjunctive procedures did not make a significant contribution to further disinfection. At the 2nd visit, bacteria were active after the use of calcium hydroxide medication; however, they were significantly reduced after a 2nd-visit preparation. Consequently, the lowest bacterial levels were found at the end of the treatment. This clinical trial, which used an rRNA and rDNA combined approach, confirmed previous studies showing that root canal preparation represents the main strategy for root canal disinfection.

scholarly journals Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of Parvovirus B19 DNA Using a Microplate Luminometer

1999 ◽  
Vol 45 (9) ◽  
pp. 1391-1396 ◽  
Author(s):  
Fabiana Fini ◽  
Giorgio Gallinella ◽  
Stefano Girotti ◽  
Marialuisa Zerbini ◽  
Monica Musiani
Keyword(s):  
Detection Limit ◽  
Parvovirus B19 ◽  
Quantitative Detection ◽  
Specific Method ◽  
Competitive Pcr ◽  
Serum Samples ◽  
Titration Curves ◽  
Viral Dnas ◽  
Assessment And Monitoring ◽  
Detection And Quantification

Abstract Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.

scholarly journals Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

2011 ◽  
Vol 34 (9) ◽  
pp. 1011-1019 ◽  
Author(s):  
Virginia García-Cañas ◽  
Monica Mondello ◽  
Alejandro Cifuentes
Keyword(s):  
Gel Electrophoresis ◽  
Dna Probes ◽  
Capillary Gel Electrophoresis ◽  
Ligation Reaction

scholarly journals pCysMod: Prediction of Multiple Cysteine Modifications Based on Deep Learning Framework

2021 ◽  
Vol 9 ◽  
Author(s):  
Shihua Li ◽  
Kai Yu ◽  
Guandi Wu ◽  
Qingfeng Zhang ◽  
Panqin Wang ◽  
...  
Keyword(s):  
Deep Learning ◽  
Low Cost ◽  
Redox Homeostasis ◽  
Cysteine Modification ◽  
Biological Regulation ◽  
Post Translational Modifications ◽  
Learning Framework ◽  
Multiple Protein ◽  
Regulation Mechanisms ◽  
Deep Learning Model

Thiol groups on cysteines can undergo multiple post-translational modifications (PTMs), acting as a molecular switch to maintain redox homeostasis and regulating a series of cell signaling transductions. Identification of sophistical protein cysteine modifications is crucial for dissecting its underlying regulatory mechanism. Instead of a time-consuming and labor-intensive experimental method, various computational methods have attracted intense research interest due to their convenience and low cost. Here, we developed the first comprehensive deep learning based tool pCysMod for multiple protein cysteine modification prediction, including S-nitrosylation, S-palmitoylation, S-sulfenylation, S-sulfhydration, and S-sulfinylation. Experimentally verified cysteine sites curated from literature and sites collected by other databases and predicting tools were integrated as benchmark dataset. Several protein sequence features were extracted and united into a deep learning model, and the hyperparameters were optimized by particle swarm optimization algorithms. Cross-validations indicated our model showed excellent robustness and outperformed existing tools, which was able to achieve an average AUC of 0.793, 0.807, 0.796, 0.793, and 0.876 for S-nitrosylation, S-palmitoylation, S-sulfenylation, S-sulfhydration, and S-sulfinylation, demonstrating pCysMod was stable and suitable for protein cysteine modification prediction. Besides, we constructed a comprehensive protein cysteine modification prediction web server based on this model to benefit the researches finding the potential modification sites of their interested proteins, which could be accessed at http://pcysmod.omicsbio.info. This work will undoubtedly greatly promote the study of protein cysteine modification and contribute to clarifying the biological regulation mechanisms of cysteine modification within and among the cells.

In situ Hybridization with Hapten-Modified DNA Probes

2015 ◽  
pp. 17-28 ◽  
Author(s):  
A. K. Raap ◽  
R. W. Dirks ◽  
N. M. Jiwa ◽  
P. M. Nederlof ◽  
M. van der Ploeg
Keyword(s):  
In Situ Hybridization ◽  
Dna Probes ◽  
Modified Dna

scholarly journals Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova

2017 ◽  
Vol 75 (11) ◽  
pp. 2615-2621 ◽  
Author(s):  
P. Gyawali ◽  
J. P. S. Sidhu ◽  
W. Ahmed ◽  
P. Jagals ◽  
S. Toze
Keyword(s):  
Environmental Samples ◽  
Quantitative Measurement ◽  
Quantitative Polymerase Chain Reaction ◽  
Detection Methods ◽  
Quantitative Detection ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Vital Stain ◽  
Polymerase Chain ◽  
Hookworm Ova

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

The rational specimen for the quantitative detection of Epstein-Barr virus DNA load

2019 ◽  
Vol 57 (5) ◽  
pp. 759-765 ◽  
Author(s):  
Wang Kedi ◽  
Xu Dongjiang ◽  
Lv Zhi ◽  
Gao Yan ◽  
Jia Kun ◽  
...  
Keyword(s):  
Viral Load ◽  
Epstein Barr Virus ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Value ◽  
Quantitative Detection ◽  
Peripheral Blood Mononuclear ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr

Abstract Background Epstein-Barr virus (EBV) DNA load monitoring in blood is essential for the diagnosis of EBV-associated diseases. However, the best-suited blood compartment for detection is still under discussion. The aim of this study was to evaluate the diagnostic value of EBV-DNA load in peripheral blood mononuclear cells (PBMC), plasma and whole blood (WB) samples. Methods A total of 156 patients, including 45 patients with infectious mononucleosis (IM), 57 patients with EBV-associated hemophagocytic lymphohistiocytosis (HLH) and 54 patients with post-transplant lymphoproliferative disorders (PTLD), were enrolled in this study. The EBV-DNA load in PBMC, plasma and WB samples were measured with real-time quantitative polymerase chain reaction (PCR). Results EBV-DNA load of patients with HLH showed no statistical difference in PBMC, plasma and WB samples, while patients with IM and PTLD showed a higher viral load in PBMC samples. The strongest correlation of EBV-DNA level was found between PBMC and WB samples among patients with IM, HLH and PTLD. The follow-up of EBV-DNA showed that the viral load became negative along with the recovery from the disease, while that in WB and PBMC would remain positive for a long time. Conclusions For the diagnosis and monitoring of EBV-DNA, the type of specimen should be chosen reasonably according to the disease. As for IM and HLH, plasma is recommended to quantify the EBV-DNA load, while PBMC and plasma are preferred in PTLD.

Sign in / Sign up

Export Citation Format

Share Document