Direct sequence confirmation of qPCR products for gene doping assay validation in horses.
gene doping
genetics
horse
Journal
Drug testing and analysis
ISSN: 1942-7611
Titre abrégé: Drug Test Anal
Pays: England
ID NLM: 101483449
Informations de publication
Date de publication:
Jun 2022
Jun 2022
Historique:
revised:
15
12
2021
received:
29
10
2021
accepted:
22
12
2021
pubmed:
8
1
2022
medline:
15
6
2022
entrez:
7
1
2022
Statut:
ppublish
Résumé
The misuse of gene therapy by the introduction of transgenes via plasmid or viral vectors as a doping agent is an increasing concern in human and animal sports, not only in consideration to fair competition but also in potential detrimental effects to welfare. Doping events can be detected by polymerase chain reaction (PCR) amplification of a transgene-specific region of DNA. Quantitative real-time PCR (qPCR) is particularly suited to confirmatory investigations where precise limits of detection can be calculated. To fully validate a qPCR experiment, it is highly desirable to confirm the identity of the amplicon. Although post-PCR techniques such as melt curve and fragment size analysis can provide strong evidence that the amplicon is as expected, sequence identity confirmation may be beneficial as part of regulatory proceedings. We present here our investigation into two alternative processes for the direct assessment of qPCR products for five genes using next-generation sequencing: ligation of sequence-ready adapters to qPCR products and qPCR assays performed with primers tailed with Illumina flow cell binding sites. To fully test the robustness of the techniques at concentrations required for gene doping detection, we also calculated a putative limit of detection for the assays. Both ligated adapters and tailed primers were successful in producing sequence data for the qPCR products without further amplification. Ligated adapters are preferred, however, as they do not require re-optimisation of existing qPCR assays.
Substances chimiques
DNA Primers
0
DNA
9007-49-2
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1017-1025Subventions
Organisme : British Horseracing Authority
Informations de copyright
© 2022 John Wiley & Sons, Ltd.
Références
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