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.

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.

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 Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction

1992 ◽  
Vol 288 (3) ◽  
pp. 1061-1067 ◽  
Author(s):  
D O Somers ◽  
S M Medd ◽  
J E Walker ◽  
M J Adams
Keyword(s):  
Polymerase Chain Reaction ◽  
Protein Sequence ◽  
Direct Analysis ◽  
Pentose Phosphate ◽  
Cdna Clones ◽  
Mature Protein ◽  
Chain Reaction ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Polymerase Chain

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters

1992 ◽  
Vol 38 (5) ◽  
pp. 365-369 ◽  
Author(s):  
Gary A. Toranzos ◽  
Abdiel J. Alvarez
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Culture Media ◽  
Direct Detection ◽  
Bacterial Cells ◽  
Chain Reaction ◽  
Detection And Identification ◽  
Low Concentrations ◽  
Polymerase Chain

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air. Key words: polymerase chain reaction, waterborne pathogens, water.

scholarly journals Identification of Streptococcus agalactiae isolated from pregnant women by 16srRNA gene

2019 ◽  
Vol 10 (2) ◽  
pp. 1523-1526
Author(s):  
Haneen Kadhum Abdul-Hamza ◽  
Ghaidaa Jihadi Mohammed
Keyword(s):  
Polymerase Chain Reaction ◽  
Streptococcus Agalactiae ◽  
Morphological Characteristics ◽  
Bacterial Cells ◽  
Chain Reaction ◽  
Vaginal Swabs ◽  
Rectal Swabs ◽  
Vitek 2 ◽  
Polymerase Chain ◽  
16Srrna Gene

The main goal of the current study is to isolate and diagnose of Streptococcus agalactiae by using the diagnostic 16 SrRNA gene. S.agalactiae was isolated from 850 samples including (425) vaginal swabs, (425) rectal swabs and identified by studying the morphological characteristics of colonies on culture, microscopic characteristics of bacterial cells, biochemical methods, Vitek 2 system, API-20 Strep, and then confirmed the identification by detection of 16SrRNA gene by Polymerase Chain reaction (PCR) followed by DNA Sequence analysis for this gene. A total of 16 isolates of S.agalactiae were isolated from 12 (2.82%) vaginal swabs, 4 (0.99%) rectal swabs, and 16SrRNA was detected in 100% of the S.agalactiae isolates.

scholarly journals Duplex Real-Time Polymerase Chain Reaction Reveals Competition Between Erwinia amylovora and E. pyrifoliae on Pear Blossoms

2008 ◽  
Vol 98 (6) ◽  
pp. 673-679 ◽  
Author(s):  
Susan M. Lehman ◽  
Won-Sik Kim ◽  
Alan J. Castle ◽  
Antonet M. Svircev
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Erwinia Amylovora ◽  
Single Species ◽  
Bacterial Cells ◽  
Chain Reaction ◽  
Causative Agents ◽  
Population Sizes ◽  
Polymerase Chain ◽  
Erwinia Species

Erwinia amylovora and E. pyrifoliae are the causative agents of fire blight and Asian pear blight, respectively. The pathogens are closely related, with overlapping host ranges. Data are unavailable on the current distribution of E. pyrifoliae and on the interaction between the two species when they are present together on the same host. In this study, a duplex real-time polymerase chain reaction (PCR) protocol was developed to monitor the population dynamics of E. amylovora and E. pyrifoliae on the surface of Bartlett pear blossoms. Bacterial cells washed from blossoms were used directly as the PCR template without DNA extraction. Primers and a probe based on the E. amylovora levansucrase gene detected all E. amylovora strains. All E. pyrifoliae strains, including the Japanese Erwinia strains previously described as E. amylovora, were detected with a primer and probe combination based on the E. pyrifoliae hrpW gene. Disease development and severity were not significantly different in blossoms inoculated with individual Erwinia species or with a mixture of the two species. However, E. amylovora grew to greater population sizes than did E. pyrifoliae in both single species inoculations and in mixtures, suggesting that E. amylovora has a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.

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