| A quick one-tube nested PCR-protocol for EPO transgene detection. | |
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MedLine Citation:
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PMID: 22539489 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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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. Copyright © 2012 John Wiley & Sons, Ltd. |
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Authors:
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Dirk A Moser; Elmo W I Neuberger; Perikles Simon |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-4-26 |
Journal Detail:
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Title: Drug testing and analysis Volume: - ISSN: 1942-7611 ISO Abbreviation: - Publication Date: 2012 Apr |
Date Detail:
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Created Date: 2012-4-27 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101483449 Medline TA: Drug Test Anal Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2012 John Wiley & Sons, Ltd. |
Affiliation:
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Johannes Gutenberg University-Mainz - Department of Sports Medicine, Rehabilitation and Disease Prevention, Mainz, Germany. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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