Back to Search
Start Over
Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay
- Source :
- Biotechnology and applied biochemistry. 64(4)
- Publication Year :
- 2016
-
Abstract
- Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.
Details
- ISSN :
- 14708744
- Volume :
- 64
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Biotechnology and applied biochemistry
- Accession number :
- edsair.pmid..........6f0502721b2ad8cbb8dd23e847f5d710