April 2024
·
11 Reads
·
2 Citations
Analytical Chemistry
This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.
April 2024
·
11 Reads
·
2 Citations
Analytical Chemistry
November 2023
·
18 Reads
·
3 Citations
Drug Testing and Analysis
The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti‐Doping Agency anti‐doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)‐based sample authentication with a multi‐target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA‐profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE‐product detection by MALDI‐TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high‐throughput format with an overall assay turnaround time of approximately 11 h. SNP‐based DNA typing via MALDI‐TOF MS thus represents a high throughput‐capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.
October 2023
·
56 Reads
·
10 Citations
International Journal of Molecular Sciences
Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.
November 2022
·
65 Reads
·
9 Citations
The Analyst
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.
November 2020
·
53 Reads
·
17 Citations
Analytical Chemistry
The discovery of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system as a programmable, RNA-guided endonuclease has revolutionized the utilization of gene technology. Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools for genome editing. However, these advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to achieve performance-enhancing effects in sporting competitions. This abuse is classified as gene doping, which is banned in sports according to the Prohibited List of the World Anti-Doping Agency (WADA). Therefore, there is a pressing need for an adequate analytical method to detect the misuse of the CRISPR/Cas system by athletes. Hence, the first aim accomplished with this study was the identification of the exogenous protein Cas9 from the bacterium Streptococcus pyogenes (SpCas9) in plasma samples by means of a bottom-up analytical approach via immunoaffinity purification, tryptic digestion, and subsequent detection by HPLC-HRMS/MS. A qualitative method validation was conducted with three specific peptides allowing for a limit of detection of 25 ng/mL. Additionally, it was shown that the developed method is also applicable to the detection of (illicit) gene regulation through the identification of catalytically inactive Cas9. A proof-of-concept administration study employing an in vivo mouse model revealed a detection window of SpCas9 for up to 8 h post administration, confirming the suitability of the test strategy for the analysis of authentic doping control samples.
... Therefore, automating most processes in doping testing is desirable as well. There are previous studies on the application of DBSs in doping testing, such as the targeting of steroids and other compounds [17][18][19][20], genetic polymorphisms [9,21,22], plasmids via the spike-in test [23], hEPO proteins with mutations [24], and mRNAs [25]. Although these previous studies described the affinity and future promise of DBSs in doping testing, there was only one report of an automated testing process [18]. ...
November 2023
Drug Testing and Analysis
... Psychological interventions such as mental skills training and biofeedback enhance focus and stress management (Lindsay et al., 2023). Recovery technologies like cryotherapy (Poignard et al., 2023) and Normatec systems (Gras, 2023) accelerate recovery, and genetic research explores the potential of gene editing and epigenetics in sports performance (Naumann et al., 2023). Data analytics and machine learning aid in performance analysis and injury prevention, while VR (Virtual Reality) (Richlan et al., 2023b) and AR (Augmented Reality) (Cossich et al., 2023) technologies create immersive training environments. ...
October 2023
International Journal of Molecular Sciences
... [117] The authors achieved a quantitative recovery that was independent of the samples' hematocrit with an optimized impact-assisted extraction Leuenberger et al. [118] mentioned DBS for RNA analysis, however, discussed them as biomarkers for substance misuse rather than as and amplified RNA. [119] Only few methods are reported for targeting oligonucleotide therapeutics in urine samples. The analysis of rat urine samples after treatment with two potential myostatin-inhibiting siRNAs was reported by Thomas et al. [120] These ...
November 2022
The Analyst
... For the detection of foreign transgene signals, surface plasmon resonance imaging has been tested with application potential in gene doping detection. 66 Some techniques will analyze the delivery tools or gene doping products, such as the presence of Cas or expressed proteins whose specific peptide signals could be captured with MS. 67 which could detect very low amounts of signals from known doping targets in various sample types. 73 qPCR assays for gene doping targets generally employ primers/probes that could target the exonexon junction sites present on transgene but absent from host genome for discrimination purpose. ...
November 2020
Analytical Chemistry