scholarly journals Accurate Quantification of AAV Vector Genomes by Quantitative PCR

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 601
Author(s):  
Cristina Martinez-Fernandez de la Camara ◽  
Michelle McClements ◽  
Robert MacLaren
Keyword(s):  
Quantitative Pcr ◽  
Enzymatic Digestion ◽  
Absolute Quantification ◽  
Proteinase K ◽  
Aav Vector ◽  
Good Laboratory ◽  
Reference Plasmid ◽  
Pre Treatment ◽  
Dna Template ◽  
Accurate Quantification

The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario.

Quantification of Biological Resistance: Introducing a Unifying Unit and Scale of Resistance

2018 ◽  
Author(s):  
Rudolf Fullybright
Keyword(s):  
Drug Resistance ◽  
Living Organism ◽  
Pathogen Resistance ◽  
Absolute Quantification ◽  
Biological Resistance ◽  
The Third ◽  
Exact Measurement ◽  
Unit Function ◽  
Third Law ◽  
Accurate Quantification

Accurate quantification of biological resistance has been impossible so far. Among the various forms of biological resistance which exist in nature, pathogen resistance to drugs is a familiar one. However, as in the case of other forms of resistance, accurately quantifying drug resistance in pathogens has been impossible up to now. Here, we introduce a mathematically-defined and uniform procedure for the absolute quantification of biological resistance deployed by any living organism in the biological realm, including and beyond drug resistance in medicine. The scheme introduced makes possible the exact measurement or computation of the extent to which resistance is deployed by any living organism regardless of kingdom and regardless of the mechanism of resistance involved. Furthermore, the Second Law of Resistance indicating that resistance has the potential to increase to infinite levels, and the Third Law of Resistance indicating that resistance comes to an end once interaction stops, the resistance unit function introduced here is fully compatible with both the Second and Third Laws of Resistance.

scholarly journals Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

Water ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3507
Author(s):  
Mark A. Ibekwe ◽  
Shelton E. Murinda ◽  
Stanley Park ◽  
Amarachukwu Obayiuwana ◽  
Marcia A. Murry ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Point Of Care ◽  
Foodborne Pathogen ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
High Rate ◽  
E Coli

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

An improved immunocytochemical staining method for large semi-thin plastic epon sections: application to GABA in rat cerebral cortex.

1993 ◽  
Vol 41 (8) ◽  
pp. 1259-1265 ◽  
Author(s):  
H J Romijn ◽  
A W Janszen ◽  
C W Pool ◽  
R M Buijs
Keyword(s):  
Staining Method ◽  
Aminobutyric Acid ◽  
Proteinase K ◽  
Immunocytochemical Staining ◽  
Staining Procedure ◽  
Gamma Aminobutyric Acid ◽  
Crucial Step ◽  
Pre Treatment ◽  
Thin Plastic ◽  
Rat Brain Tissue

We describe here a procedure that significantly enhances the intensity and method-specificity of immunocytochemical staining in large mounted semi-thin plastic Epon sections. The procedure was developed for the detection of the neurotransmitter gamma-aminobutyric acid (GABA) in rat brain tissue fixed with glutaraldehyde, but it may also be helpful in unmasking other antigens under different conditions. In addition to some practical suggestions for improving the reproducibility of the staining procedure, we demonstrate that the crucial step in the procedure is pre-treatment of the deplasticized sections with proteinase-K before exposure to the first antibody. This leads to a high morphological resolution and an excellent immunocytochemical signal.

scholarly journals HILIC-Enabled 13C Metabolomics Strategies: Comparing Quantitative Precision and Spectral Accuracy of QTOF High- and QQQ Low-Resolution Mass Spectrometry

Metabolites ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 63 ◽  
Author(s):  
André Feith ◽  
Attila Teleki ◽  
Michaela Graf ◽  
Lorenzo Favilli ◽  
Ralf Takors
Keyword(s):  
Mass Spectrometry ◽  
Isotope Dilution Mass Spectrometry ◽  
Absolute Quantification ◽  
Spectral Accuracy ◽  
Time Resolved ◽  
13C Tracer ◽  
Accurate Quantification ◽  
Tracer Experiments

Dynamic 13C-tracer-based flux analyses of in vivo reaction networks still require a continuous development of advanced quantification methods applying state-of-the-art mass spectrometry platforms. Utilizing alkaline HILIC chromatography, we adapt strategies for a systematic quantification study in non- and 13C-labeled multicomponent endogenous Corynebacterium glutamicum extracts by LC-QTOF high resolution (HRMS) and LC-QQQ tandem mass spectrometry (MS/MS). Without prior derivatization, a representative cross-section of 17 central carbon and anabolic key intermediates were analyzed with high selectivity and sensitivity under optimized ESI-MS settings. In column detection limits for the absolute quantification range were between 6.8–304.7 (QQQ) and 28.7–881.5 fmol (QTOF) with comparable linearities (3–5 orders of magnitude) and enhanced precision using QQQ-MRM detection. Tailor-made preparations of uniformly (U)13C-labeled cultivation extracts for isotope dilution mass spectrometry enabled the accurate quantification in complex sample matrices and extended linearities without effect on method parameters. Furthermore, evaluation of metabolite-specific m+1-to-m+0 ratios (ISR1:0) in non-labeled extracts exhibited sufficient methodical spectral accuracies with mean deviations of 3.89 ± 3.54% (QTOF) and 4.01 ± 3.01% (QQQ). Based on the excellent HILIC performance, conformity analysis of time-resolved isotopic enrichments in 13C-tracer experiments revealed sufficient spectral accuracy for QQQ-SIM detection. However, only QTOF-HRMS ensures determination of the full isotopologue space in complex matrices without mass interferences.

miRNA analysis between malignant and benign tissue and circulating exosomes (CE) in patients (pts) with tongue squamous cell carcinoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 6088-6088
Author(s):  
Guilherme Rabinowits ◽  
Ludmila Flores ◽  
Anne M. O'Neill ◽  
Kristen Stevenson ◽  
Irene M. Ghobrial ◽  
...  
Keyword(s):  
Tumor Tissue ◽  
Tongue Cancer ◽  
Complete Response ◽  
Tumor Location ◽  
Proteinase K ◽  
Benign Tissue ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Pre Treatment ◽  
Nanostring Technology

6088 Background: The identification of a specific miRNA pattern in HNSCC is challenging given the heterogeneity of this disease and different methodologies used in prior studies. Here we chose to group pts according to primary tumor location and used Nanostring as a larger miRNA platform. Methods: CE-miRNA was isolated from 500ul of plasma collected in Heparin/EDTA tubes. Samples were pretreated with Pacific Hemostasis Thromboplastin D. Supernatant was treated with ExoQuick. Exosomal pellet was resuspended in 1ml of QIAzol Lysis Reagent. Formalin fixed paraffin embedded (FFPE) samples were deparaffinized and digested with proteinase K. miRNA from plasma and FFPE were isolated with miRNeasy Mini Kit. Samples were processed by Nanostring Technology. In this analysis, only tongue cancer pts were selected due to availability of paired tumor and benign tissue miRNA in conjunction with CE from the same pts and others with tongue cancer. Results: 21 HNSCC pts and 32 age-matched controls have enrolled to date. For homogeneity we analyzed only patients with tongue cancer (n=9): 8 males, median age 55.5 years (range 24-64), all had stage IVA tongue cancer (6 base of tongue), 6 HPV/p16 positive. Nine pts received chemoradiation; 4 also had surgery; 8 had clinical and radiological complete response. Of the 800 miRNAs tested 62 were overexpressed and 15 were suppressed in the tumor compared to benign tissue in the same pts. Of those overexpressed and suppressed in tumor tissue, 4 (miR23a-3p; miR150-5p; miR199a-5p; miR203) had a post/pre treatment average ratio (PPTAR) less than 1, while 4 (miR720; miR1253; miR145-5p; miR1283) had PPTAR greater than 1, respectively on CE of pts. miR150-5p was suppressed and miR1283, miR145-5p and miR1253 were overexpressed on CE of control samples compared to pre treatment samples of pts. Conclusions: Of the 800 miRNAs, 8 appear to correlate with treatment response. The average levels of 4 of those miRNA mirror the average levels of tumor tissue miRNA and is in an inverse relationship with benign tissue and control blood samples, suggesting this may be a unique signature for tongue cancer. This observation needs to be validated in larger studies.

scholarly journals Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

2012 ◽  
Vol 78 (12) ◽  
pp. 4481-4489 ◽  
Author(s):  
Robert Brankatschk ◽  
Natacha Bodenhausen ◽  
Josef Zeyer ◽  
Helmut Bürgmann
Keyword(s):  
Microbial Community ◽  
Quantitative Pcr ◽  
Environmental Samples ◽  
Absolute Quantification ◽  
Geobacter Sulfurreducens ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Nostoc Commune ◽  
Content Type

ABSTRACTReal-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting thenifHand 16S rRNA genes. TheEvalues of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in theirEvalues, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences inEand was derived from the ΔΔCTmethod with correction forE, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures fromGeobacter sulfurreducens(DSM 12127) andNostoc commune(Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in theirEvalues. While the SC method deviated from the expectednifHgene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences inEbetween the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

Distinguishing Bacteriolytic from Bacteriostatic Activity of an Antibiotic Agent by Real-Time Quantitative PCR

2011 ◽  
Vol 343-344 ◽  
pp. 357-360
Author(s):  
Zhi Ping Wang
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Bacteriostatic Activity ◽  
Antibiotic Agent ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Real Time Quantitative Pcr ◽  
Pcr Product ◽  
Dna Template ◽  
Quantitative Pcr Assay

A simple and rapid real-time quantitative PCR assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. Bacteria suspension was incubated with the compound solution, the mixture was centrifuged and supernatant was removed completely, the obtained pellet was then used as the DNA template for PCR. Then the bacteriostatic and bacteriolytic activity can be inferred by the quantity of PCR product. Moreover, the parameters that influence the assay sensitivity was discussed.

scholarly journals Complete enzymatic digestion of double-stranded RNA to nucleosides enables accurate quantification of dsRNA

2021 ◽  
Author(s):  
Steven R. Strezsak ◽  
Penny J. Beuning ◽  
Nicholas J. Skizim
Keyword(s):  
Rna Interference ◽  
Rapid Growth ◽  
Enzymatic Digestion ◽  
Robust Method ◽  
Double Stranded Rna ◽  
Accurate Quantification

The rapid growth of research focusing on RNA, especially for RNA interference applications, has created a need for a robust method that can accurately determine the concentration of long dsRNA.

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