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Article ; Online: A brief history of the development of a gene doping test.

Baoutina, Anna

Bioanalysis

2020 Volume 12, Issue 11, Page(s) 723–727

MeSH term(s)	Athletes ; Doping in Sports ; Genetic Testing ; Humans ; Substance Abuse Detection
Language	English
Publishing date	2020-04-23
Publishing country	England
Document type	Editorial
ISSN	1757-6199
ISSN (online)	1757-6199
DOI	10.4155/bio-2020-0056
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Article ; Online: Novel design of nucleic acid standards for hydrolysis probe-based PCR with melting analysis.

Baoutina, Anna / Bhat, Somanath

Gene therapy

2021 Volume 29, Issue 7-8, Page(s) 425–430

Abstract: Probe-based polymerase chain reaction (PCR), a popular method in genetic testing, could be prone to false positive or positively biased results if standards used for positive controls or as calibrants accidentally contaminate samples during analysis. To ... ...

Abstract	Probe-based polymerase chain reaction (PCR), a popular method in genetic testing, could be prone to false positive or positively biased results if standards used for positive controls or as calibrants accidentally contaminate samples during analysis. To prevent this, a unique design strategy for nucleic acid standards has been developed. Several in-house designed synthetic standards and corresponding test targets were analysed in specific probe-based PCR assays in the presence of SYTO 82™, an intercalating dye compatible with a probe-labelling FAM (6-Carboxyfluorescein) fluorophore. PCR was followed by melting and fragment size analyses. We showed that a standard can be designed to allow discrimination from the test target in post-PCR melting analysis based on differences in melting temperature (Tm). A good predictor of Tm differences for the paired amplicons was the software package uMelt, but not the length of the amplicons nor guanine-cytosine (GC) content. Tm-based determination can be complimented by electrophoresis to measure differences in amplicons' length. Designing genetic standards using the described method for tests that utilise probe-based PCR will prevent false positive and inaccurate results, while also simplifying the test and reducing its cost.
MeSH term(s)	Fluorescent Dyes ; Hydrolysis ; Nucleic Acids/genetics ; Polymerase Chain Reaction/methods ; Temperature
Chemical Substances	Fluorescent Dyes ; Nucleic Acids
Language	English
Publishing date	2021-08-19
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1191036-7
ISSN	1476-5462 ; 0969-7128
ISSN (online)	1476-5462
ISSN	0969-7128
DOI	10.1038/s41434-021-00288-0
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Article ; Online: PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.

Wilkin, Tessa / Hamilton, Natasha A / Cawley, Adam T / Bhat, Somanath / Baoutina, Anna

International journal of molecular sciences

2024 Volume 25, Issue 5

Abstract: The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: ...

Abstract	The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping:
MeSH term(s)	Animals ; Horses/genetics ; Erythropoietin/genetics ; Australia ; Plasmids ; DNA/genetics ; Real-Time Polymerase Chain Reaction
Chemical Substances	Erythropoietin (11096-26-7) ; DNA (9007-49-2)
Language	English
Publishing date	2024-02-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms25052570
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Article ; Online: Towards a robust test to detect gene doping for anabolic enhancement in human athletes.

Baoutina, Anna / Bhat, Somanath / Li, Desmond K / Emslie, Kerry R

Drug testing and analysis

2022 Volume 15, Issue 3, Page(s) 314–323

Abstract: Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we ... ...

Abstract	Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone-releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.
MeSH term(s)	Humans ; Athletes ; Transgenes ; Polymerase Chain Reaction/methods ; Genetic Therapy ; DNA ; Doping in Sports
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2022-12-18
Publishing country	England
Document type	Journal Article
ZDB-ID	2462336-2
ISSN	1942-7611 ; 1942-7603
ISSN (online)	1942-7611
ISSN	1942-7603
DOI	10.1002/dta.3411
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Article ; Online: Storage Stability of Solutions of DNA Standards.

Baoutina, Anna / Bhat, Somanath / Partis, Lina / Emslie, Kerry R

Analytical chemistry

2019 Volume 91, Issue 19, Page(s) 12268–12274

Abstract: High accuracy, reliability, and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally ... ...

Abstract	High accuracy, reliability, and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally important. In this study, we investigated the stability of dilute (4000 cp/μL, nominal concentration, equivalent to 0.02 ng/mL) DNA solutions stored at 4, -20, and -80 °C in the presence or absence of nucleic acid carriers. As representative examples, we used different formulations of a linearized plasmid DNA solution considered for characterization as reference materials (RMs) for specific applications. Employing droplet digital PCR, a highly accurate and precise method for quantification of nucleic acid not requiring a calibrant, we demonstrated that inclusion of a carrier nucleic acid in the formulation (at 50 ng/μL) improved the plasmid stability at -20 and -80 °C. For the case of a DNA standard used in real-time PCR assays for human erythropoietin gene, cDNA or transcript, we found that inclusion of yeast RNA in the formulation was preferred over salmon testes DNA as it had no effect on PCR amplification and provided the lowest relative expanded uncertainty for the characterized RM. RNA background may also be preferred as it is applicable to a broader range of DNA RMs. Our findings are important in production of reliable, stable DNA standards, including DNA RMs. These results can be used when selecting protocols for stable storage of DNA either extracted from biological samples or synthesized in a laboratory.
MeSH term(s)	Animals ; DNA/chemistry ; DNA/standards ; Erythropoietin/genetics ; Freezing ; Humans ; Plasmids ; Quality Control ; Real-Time Polymerase Chain Reaction/standards ; Reference Standards ; Reproducibility of Results ; Salmon/genetics ; Temperature
Chemical Substances	EPO protein, human ; Erythropoietin (11096-26-7) ; DNA (9007-49-2)
Language	English
Publishing date	2019-09-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.9b02334
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Article ; Online: Equine performance genes and the future of doping in horseracing.

Wilkin, Tessa / Baoutina, Anna / Hamilton, Natasha

Drug testing and analysis

2017 Volume 9, Issue 9, Page(s) 1456–1471

Abstract: A horse's success on the racetrack is determined by genetics, training and nutrition, and their translation into physical traits such as speed, endurance and muscle strength. Advances in genetic technologies are slowly explaining the roles of specific ... ...

Abstract	A horse's success on the racetrack is determined by genetics, training and nutrition, and their translation into physical traits such as speed, endurance and muscle strength. Advances in genetic technologies are slowly explaining the roles of specific genes in equine performance, and offering new insights into the development of novel therapies for diseases and musculoskeletal injuries that cause early retirement of many racehorses. Gene therapy approaches may also soon provide new means to artificially enhance the physical performance of racehorses. Gene doping, the misuse of gene therapies for performance enhancement, is predicted to be the next phase of doping faced by horseracing. The risk of gene doping to human sports has been recognised for almost 15 years, and the introduction of the first gene doping detection tests for doping control in human athletes is imminent. Gene doping is also a threat to horseracing, but there are currently no methods to detect it. Efficient and accurate detection methods need to be developed to deter those looking to use gene doping in horses and to maintain the integrity of the sport. Methods developed for human athletes could offer an avenue for detection in racehorses. Development of an equine equivalent test will first require identification of equine genes that will likely be targeted by gene doping attempts. This review focuses on genes that have been linked to athletic performance in horses and, therefore, could be targeted for genetic manipulation. The risks associated with gene doping and approaches to detect gene doping are also discussed. Copyright © 2017 John Wiley & Sons, Ltd.
MeSH term(s)	Animals ; Athletic Performance ; Doping in Sports/methods ; Horses ; Humans
Language	English
Publishing date	2017-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2462336-2
ISSN	1942-7611 ; 1942-7603
ISSN (online)	1942-7611
ISSN	1942-7603
DOI	10.1002/dta.2198
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Article: Storage Stability of Solutions of DNA Standards

Baoutina, Anna / Bhat, Somanath / Partis, Lina / Emslie, Kerry R

Analytical chemistry. 2019 Aug. 29, v. 91, no. 19

2019

Abstract	High accuracy, reliability, and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally important. In this study, we investigated the stability of dilute (4000 cp/μL, nominal concentration, equivalent to 0.02 ng/mL) DNA solutions stored at 4, −20, and −80 °C in the presence or absence of nucleic acid carriers. As representative examples, we used different formulations of a linearized plasmid DNA solution considered for characterization as reference materials (RMs) for specific applications. Employing droplet digital PCR, a highly accurate and precise method for quantification of nucleic acid not requiring a calibrant, we demonstrated that inclusion of a carrier nucleic acid in the formulation (at 50 ng/μL) improved the plasmid stability at −20 and −80 °C. For the case of a DNA standard used in real-time PCR assays for human erythropoietin gene, cDNA or transcript, we found that inclusion of yeast RNA in the formulation was preferred over salmon testes DNA as it had no effect on PCR amplification and provided the lowest relative expanded uncertainty for the characterized RM. RNA background may also be preferred as it is applicable to a broader range of DNA RMs. Our findings are important in production of reliable, stable DNA standards, including DNA RMs. These results can be used when selecting protocols for stable storage of DNA either extracted from biological samples or synthesized in a laboratory.
Keywords	complementary DNA ; droplets ; erythropoietin ; genes ; genetic analysis ; humans ; messenger RNA ; plasmids ; quantitative polymerase chain reaction ; salmon ; storage quality ; testes ; uncertainty ; yeasts
Language	English
Dates of publication	2019-0829
Size	p. 12268-12274.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.9b02334
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Article ; Online: Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.

Baoutina, A / Bhat, S / Zheng, M / Partis, L / Dobeson, M / Alexander, I E / Emslie, K R

Gene therapy

2016 Volume 23, Issue 10, Page(s) 708–717

Abstract: There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression ... ...

Abstract	There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.
MeSH term(s)	DNA, Recombinant/genetics ; DNA, Recombinant/metabolism ; Erythropoietin/genetics ; Erythropoietin/metabolism ; Genetic Therapy/standards ; Humans ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Messenger/standards ; Reference Standards ; Transgenes
Chemical Substances	DNA, Recombinant ; RNA, Messenger ; Erythropoietin (11096-26-7)
Language	English
Publishing date	2016-10
Publishing country	England
Document type	Journal Article
ZDB-ID	1191036-7
ISSN	1476-5462 ; 0969-7128
ISSN (online)	1476-5462
ISSN	0969-7128
DOI	10.1038/gt.2016.47
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Article ; Online: Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system.

Baoutina, A / Coldham, T / Bains, G S / Emslie, K R

Gene therapy

2010 Volume 17, Issue 8, Page(s) 1022–1032

Abstract: As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major ... ...

Abstract	As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.
MeSH term(s)	Athletes ; Athletic Performance ; Doping in Sports/prevention & control ; Erythropoietin/genetics ; Gene Transfer Techniques ; Genetic Therapy ; Humans ; Polymerase Chain Reaction/methods ; Reproducibility of Results ; Transgenes
Chemical Substances	Erythropoietin (11096-26-7)
Language	English
Publishing date	2010-08
Publishing country	England
Document type	Evaluation Studies ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1191036-7
ISSN	1476-5462 ; 0969-7128
ISSN (online)	1476-5462
ISSN	0969-7128
DOI	10.1038/gt.2010.49
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Article: alpha-Tocopherol in atherogenesis: do we know its real role?

Neuzil, J / Baoutina, A

Cardiovascular drugs and therapy

1998 Volume 12, Issue 5, Page(s) 421–423

MeSH term(s)	Antioxidants/therapeutic use ; Arteriosclerosis/drug therapy ; Dietary Supplements ; Humans ; Vitamin E/therapeutic use
Chemical Substances	Antioxidants ; Vitamin E (1406-18-4)
Language	English
Publishing date	1998-10
Publishing country	United States
Document type	Editorial ; Review
ZDB-ID	639068-7
ISSN	1573-7241 ; 0920-3206
ISSN (online)	1573-7241
ISSN	0920-3206
DOI	10.1023/a:1007782012646
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