A quick one-tube nested PCR-protocol for EPO transgene detection
- Publication type:
- Journal article
- Metadata:
-
- Autoren
- Dirk A Moser
- Neuberger E. W.
- Perikles Simon
- Sammlungen
- metadata
- ISSN
- 1942-7611
- Ausgabe der Veröffentlichung
- 11
- Zeitschrift
- Drug testing and analysis
- Schlüsselwörter
- 796 Sport
- 796 Athletic and outdoor sports and games
- Sprache
- eng
- Paginierung
- Seiten: 870 - 875
- Datum der Veröffentlichung
- 2012
- Herausgeber
- Wiley
- Herausgeber URL
- http://dx.doi.org/10.1002/dta.1348
- Datum der Datenerfassung
- 2020
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2020
- Zugang
- Public
- Titel
- A quick one-tube nested PCR-protocol for EPO transgene detection
- Ausgabe der Zeitschrift
- 4
Data source: METADATA.UB
- Other metadata sources:
-
- Autoren
- Dirk A Moser
- Elmo WI Neuberger
- Perikles Simon
- Autoren-URL
- https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000311436000009&DestLinkType=FullRecord&DestApp=WOS_CPL
- DOI
- 10.1002/dta.1348
- Externe Identifier
- Clarivate Analytics Document Solution ID: 041XT
- PubMed Identifier: 22539489
- ISSN
- 1942-7603
- Ausgabe der Veröffentlichung
- 11
- Zeitschrift
- DRUG TESTING AND ANALYSIS
- Schlüsselwörter
- erythropoietin (EPO)
- one-tube nested PCR (OTN PCR)
- gene doping
- SpiPCR
- antisense PCR
- Paginierung
- 870 - 875
- Datum der Veröffentlichung
- 2012
- Status
- Published
- Titel
- A quick one-tube nested PCR-protocol for EPO transgene detection
- Sub types
- Article
- Ausgabe der Zeitschrift
- 4
Data source: Web of Science (Lite)
- Abstract
- <jats:p>The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore very important to develop valid detection techniques for transgenic DNA (tDNA) with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with sufficiently high sensitivity. Two utilize a real‐time PCR‐based approach and one uses a primer‐internal, intron‐spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate.</jats:p><jats:p>Based on spiPCR, here we present a novel one‐tube nested PCR approach that minimizes the chances for cross‐contamination and shows increased sensitivity compared to non‐nested PCR techniques. To further reduce the occurrence of false‐positives based on cross‐contamination, a multi‐functional 19bp extended erythropoietin standard (<jats:italic>EPO</jats:italic>) was cloned which can be easily differentiated from transgenic <jats:italic>EPO</jats:italic> DNA (t<jats:italic>EPO</jats:italic>) and can be used as an internal or external positive control in PCR‐based applications.</jats:p><jats:p>We found that one‐tube nested PCR is superior in terms of sensitivity and specificity compared to conventional PCR, and shows similar sensitivity compared to real‐time based PCR assays. Although it did not reach sensitivity of spiPCR, the one‐tube nested PCR technique described here is less laborious, less expensive and much faster than spiPCR. This technique might therefore be useful as a pre‐screening tool for gene doping in the future. Copyright © 2012 John Wiley & Sons, Ltd.</jats:p>
- Autoren
- Dirk A Moser
- Elmo WI Neuberger
- Perikles Simon
- DOI
- 10.1002/dta.1348
- eISSN
- 1942-7611
- ISSN
- 1942-7603
- Ausgabe der Veröffentlichung
- 11
- Zeitschrift
- Drug Testing and Analysis
- Sprache
- en
- Online publication date
- 2012
- Paginierung
- 870 - 875
- Datum der Veröffentlichung
- 2012
- Status
- Published
- Herausgeber
- Wiley
- Herausgeber URL
- http://dx.doi.org/10.1002/dta.1348
- Datum der Datenerfassung
- 2023
- Titel
- A quick one‐tube nested PCR‐protocol for EPO transgene detection
- Ausgabe der Zeitschrift
- 4
Data source: Crossref
- Abstract
- The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore very important to develop valid detection techniques for transgenic DNA (tDNA) with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with sufficiently high sensitivity. Two utilize a real-time PCR-based approach and one uses a primer-internal, intron-spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate. Based on spiPCR, here we present a novel one-tube nested PCR approach that minimizes the chances for cross-contamination and shows increased sensitivity compared to non-nested PCR techniques. To further reduce the occurrence of false-positives based on cross-contamination, a multi-functional 19bp extended erythropoietin standard (EPO) was cloned which can be easily differentiated from transgenic EPO DNA (tEPO) and can be used as an internal or external positive control in PCR-based applications. We found that one-tube nested PCR is superior in terms of sensitivity and specificity compared to conventional PCR, and shows similar sensitivity compared to real-time based PCR assays. Although it did not reach sensitivity of spiPCR, the one-tube nested PCR technique described here is less laborious, less expensive and much faster than spiPCR. This technique might therefore be useful as a pre-screening tool for gene doping in the future.
- Addresses
- Johannes Gutenberg University-Mainz-Department of Sports Medicine, Rehabilitation and Disease Prevention, Mainz, Germany.
- Autoren
- Dirk A Moser
- Elmo WI Neuberger
- Perikles Simon
- DOI
- 10.1002/dta.1348
- eISSN
- 1942-7611
- Externe Identifier
- PubMed Identifier: 22539489
- Open access
- false
- ISSN
- 1942-7603
- Ausgabe der Veröffentlichung
- 11
- Zeitschrift
- Drug testing and analysis
- Schlüsselwörter
- Humans
- Erythropoietin
- DNA
- Polymerase Chain Reaction
- Transgenes
- Time Factors
- Doping in Sports
- Real-Time Polymerase Chain Reaction
- Sprache
- eng
- Medium
- Print-Electronic
- Online publication date
- 2012
- Paginierung
- 870 - 875
- Datum der Veröffentlichung
- 2012
- Status
- Published
- Datum der Datenerfassung
- 2012
- Titel
- A quick one-tube nested PCR-protocol for EPO transgene detection.
- Sub types
- Research Support, Non-U.S. Gov't
- Journal Article
- Ausgabe der Zeitschrift
- 4
Data source: Europe PubMed Central
- Abstract
- The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore very important to develop valid detection techniques for transgenic DNA (tDNA) with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with sufficiently high sensitivity. Two utilize a real-time PCR-based approach and one uses a primer-internal, intron-spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate. Based on spiPCR, here we present a novel one-tube nested PCR approach that minimizes the chances for cross-contamination and shows increased sensitivity compared to non-nested PCR techniques. To further reduce the occurrence of false-positives based on cross-contamination, a multi-functional 19bp extended erythropoietin standard (EPO) was cloned which can be easily differentiated from transgenic EPO DNA (tEPO) and can be used as an internal or external positive control in PCR-based applications. We found that one-tube nested PCR is superior in terms of sensitivity and specificity compared to conventional PCR, and shows similar sensitivity compared to real-time based PCR assays. Although it did not reach sensitivity of spiPCR, the one-tube nested PCR technique described here is less laborious, less expensive and much faster than spiPCR. This technique might therefore be useful as a pre-screening tool for gene doping in the future.
- Date of acceptance
- 2012
- Autoren
- Dirk A Moser
- Elmo WI Neuberger
- Perikles Simon
- Autoren-URL
- https://www.ncbi.nlm.nih.gov/pubmed/22539489
- DOI
- 10.1002/dta.1348
- eISSN
- 1942-7611
- Ausgabe der Veröffentlichung
- 11
- Zeitschrift
- Drug Test Anal
- Schlüsselwörter
- DNA
- Doping in Sports
- Erythropoietin
- Humans
- Polymerase Chain Reaction
- Real-Time Polymerase Chain Reaction
- Time Factors
- Transgenes
- Sprache
- eng
- Country
- England
- Paginierung
- 870 - 875
- Datum der Veröffentlichung
- 2012
- Status
- Published
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2013
- Titel
- A quick one-tube nested PCR-protocol for EPO transgene detection.
- Sub types
- Journal Article
- Research Support, Non-U.S. Gov't
- Ausgabe der Zeitschrift
- 4
Data source: PubMed
- Beziehungen:
- Property of