scholarly journals Модель генерации флуоресцентного сигнала интеркаляционного красителя в ходе полимеразной цепной реакции

2020 ◽  
Vol 90 (9) ◽  
pp. 1581
Author(s):  
А.А. Федоров ◽  
Д.Г. Сочивко ◽  
Д.А. Варламов
Keyword(s):  
Polymerase Chain Reaction ◽  
Reaction Kinetics ◽  
Reaction Model ◽  
Chain Reaction ◽  
Fluorescent Reporter ◽  
Fluorescent Signal ◽  
Quantitative Estimates ◽  
Polymerase Chain ◽  
Simulation Results ◽  
Kinetics Of

Currently, a number of models of polymerase chain reaction have been proposed, claiming to be accurate quantitative estimates of the reaction results. However, all these models assume identity the kinetics of the reaction product and the used fluorescent reporter. In the present work a polymerase chain reaction model is proposed, product reaction kinetics of which are generated by intercalation dye. An analysis of the simulation results demonstrates noticeable differences between the kinetics of the reaction product and the fluorescent signal of the coupled intercalation dye.

Considerations in the development and validation of real-time quantitative polymerase chain reaction and its application in regulated bioanalysis to characterize the cellular kinetics of CAR-T products in clinical studies

Bioanalysis ◽  
2020 ◽  
Author(s):  
Tong-yuan Yang ◽  
Rajitha Doddareddy
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Method Development ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Cellular Kinetics ◽  
Car T ◽  
Polymerase Chain ◽  
Development And Validation ◽  
Kinetics Of

Real-time quantitative polymerase chain reaction (qPCR) has become the standard method for monitoring cellular kinetics of CAR-T therapies with measurement of the CAR transgene copy numbers in peripheral blood mononuclear cells isolated from patients receiving the treatment. Unlike other biophysical and immunological methodologies for bioanalytical characterization of conventional small molecule drugs or protein biologics, there is no relevant regulatory guidance to date on the method development and validation for quantitative qPCR assays employed during clinical development of CAR-T products. This paper will provide an overview and considerations in the development and validation of a qPCR assay from sample extraction to assay parameters and its implementation in regulated bioanalysis for CAR-T or other types of cell therapies.

Neural-Genetic Optimization of Temperature Control in a Continuous Flow Polymerase Chain Reaction Microdevice

2005 ◽  
Author(s):  
Hing Wah Lee ◽  
Parthiban Arunasalam ◽  
Ishak A. Azid ◽  
Kankanhally N. Seetharamu
Keyword(s):  
Polymerase Chain Reaction ◽  
Reaction Zone ◽  
Temperature Control ◽  
Continuous Flow ◽  
Microfluidic Channel ◽  
Microfluidic Channels ◽  
Genetic Optimization ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Simulation Results

In this study, a hybridized neural-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA) is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated zones of 94°C, 65°C and 72°C for the corresponding process of denaturing, annealing and extension to complete a cycle of polymerase chain reaction. Three separately addressable heaters provide heat input to each zone, microfluidic channels allow for the transport of fluid between zones and thermal isolation between the zones is maintained by machining air-gaps into the device. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of ±1°C or better irrespective of changing ambient conditions. Results obtained from the FEA simulation are compared with published experimental work. Simulation results show good comparison with experimental work for the temperature control in each reaction zone of the microfluidic channels. The data is then used to train the ANN to predict the temperature distribution of the microfluidic channel for new heater input power and fluid flow rate. Using these data, optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. The objective of the optimization is to minimize the temperature difference in each reaction zone of the microfluidic channel while satisfying the residence time requirement. Finally, the optimized results for the CPCR device are used to build a new FEA model for numerical simulation analysis. The simulation results for the neural-genetic optimized CPCR model and the initial CPCR model are then compared. The neural-genetic optimized model shows a significant improvement from the initial model establishing the optimization methods superiority.

scholarly journals Kinetics of polymerase chain reaction positivity in patients with Clostridioides difficile infection

2021 ◽  
Vol 14 ◽  
pp. 175628482110504
Author(s):  
Srishti Saha ◽  
Devvrat Yadav ◽  
Ryan Pardi ◽  
Robin Patel ◽  
Sahil Khanna ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Median Time ◽  
Median Duration ◽  
Treatment Initiation ◽  
Chain Reaction ◽  
Duration Of Treatment ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Polymerase Chain ◽  
Kinetics Of

Background: Polymerase chain reaction (PCR) is a sensitive test for diagnosing Clostridioides difficile infection (CDI) and could remain positive following resolution of CDI. The kinetics of PCR positivity following antibiotics for CDI is unknown. We studied this and whether it predicted CDI recurrence. Methods: Adults with CDI from October 2009 to May 2017 were included. Serial stool samples within 60 days of treatment were collected. Recurrent CDI was defined as diarrhea after interim symptom resolution with positive stool PCR within 56 or 90 days of treatment completion. Contingency table analysis was used to assess the risk of recurrence. Results: Fifty patients were included [median age: 51 (range = 20–86) years, 66% women]. Treatment given was metronidazole, 50% (25); vancomycin, 44% (22); both, 4% (2); and fidaxomicin, 2% (1). Median duration of treatment for all 50 patients was 14 (range = 8–60) days. The median duration of treatment in patients who got prolonged therapy (>14 days) ( n = 10) was 47 (range = 18–60) days. Median time to negative PCR was 9 (95% CI, 7–14) days from treatment initiation, which did not differ by antibiotics given ( p = 0.5). A positive PCR during or after treatment was associated with a higher risk of recurrence at 56 days ( p = 0.02) and at 90 days ( p = 0.009). Conclusion: The median time to negative PCR in CDI was 9 days from treatment initiation. The PCR positivity during or after treatment may be useful for recurrence prediction; larger studies are needed to validate these results.

scholarly journals Frequency of coexistence and kinetics of the BCR-ABL1 transcript level and allele burden of JAK2V617F and CALR Type 1, 2 gene mutations in patients with chronic myeloid leukemia

2020 ◽  
Vol 65 (3) ◽  
pp. 253-280
Author(s):  
A. O. Abdullaev ◽  
E. A. Stepanova ◽  
T. V. Makarik ◽  
E. Y. Nikulina ◽  
S. A. Treglazova ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Transcript Level ◽  
Chimeric Gene ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Kinetics Of

Introduction. The pathogenesis of myeloproliferative neoplasms is associated with the chimeric gene BCR-ABL1 or with one of the driver mutations in the genes JAK2, MPL and CALR (Calreticulin). However, the classifi cation of the World Health Organization lists no myeloid neoplasms with more than one driver genetic abnormality. Aim. To search for mutations in the genes JAK2, MPL and CALR in patients with BCR-ABL1-positive chronic myeloid leukemia (CML), as well as to evaluate the kinetics of the discovered mutations during tyrosine kinase inhibitor (TKI) therapy. Materials and methods. mRNA and DNA samples isolated from blood and bone marrow cells of 567 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2019 period were included in the study The BCR-ABL1 transcript level was determined using a highly sensitive quantitative real-time polymerase chain reaction. The mutations JAK2V617F and MPLW515L/K were detected using real-time quantitative allele-specifi c polymerase chain reaction. Mutations in the CALR gene were investigated using fragment analysis followed by Sanger sequencing. Results. The combination of the BCR-ABL1, JAK2 and CALR gene mutations among CML patients receiving TKIs was 1.23 % (7/567). Out of these, the combination of BCR-ABL1 with JAK2V617F and the combination of BCR-ABL1 with CALR gene mutations were detected in 0.88 % (5/567) and 0.35 % (2/567) of cases, respectively. During TKI therapy, in 5 out of 7 patients, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these patients, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second-generation TKI preparations. Conclusions. The combination of the BCR-ABL1 chimeric gene with gene mutations Jak2 or CALR was a rare event and amounted to 0.88 and 0.35 % of cases, respectively. The combination of BCR-ABL1 with Jak2V617F and CALR mutations does not always impede the achievement of major MR.

scholarly journals A Colorimetric Assay to Quantify Dehydrogenase Activity in Crude Cell Lysates

2002 ◽  
Vol 7 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Kimberly M. Mayer ◽  
Frances H. Arnold
Keyword(s):  
Polymerase Chain Reaction ◽  
Dehydrogenase Activity ◽  
Colorimetric Assay ◽  
Bacterial Cells ◽  
Phenazine Methosulfate ◽  
Chain Reaction ◽  
Phosphogluconate Dehydrogenase ◽  
Polymerase Chain ◽  
Kinetics Of ◽  
Purple Formazan

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7°C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

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