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Biotechnol Appl Biochem. 2017 Jul;64(4):549-554. doi: 10.1002/bab.1518. Epub 2017 Mar 23.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Author information

1
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.
2
University of Chinese Academy of Sciences, Beijing, People's Republic of China.
3
College of Physical Education, Soochow University, Suzhou, People's Republic of China.

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.

KEYWORDS:

IGF-I; gene doping; gene therapy; internal reference control (IRC); real-time quantitative PCR; recombinant adeno-associated virus (rAAV)

PMID:
27301870
DOI:
10.1002/bab.1518
[Indexed for MEDLINE]

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