A quick one-tube nested PCR-protocol for EPO transgene detection

Publikationstyp:
Zeitschriftenaufsatz
Metadaten:
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

Datenquelle: METADATA.UB

Andere Metadatenquellen:
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

Datenquelle: 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 &amp; 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

Datenquelle: 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

Datenquelle: 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

Datenquelle: PubMed

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