Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.
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.
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.|University of Chinese Academy of Sciences, Beijing, People's Republic of China.
Jin-Ju
JJ
Zhang
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.|College of Physical Education, Soochow University, Suzhou, People's Republic of China.
Jing-Feng
JF
Xu
College of Physical Education, Soochow University, Suzhou, People's Republic of China.
Yong-Wei
YW
Shen
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.|College of Physical Education, Soochow University, Suzhou, People's Republic of China.
Shi-Jiao
SJ
Ma
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.|College of Physical Education, Soochow University, Suzhou, People's Republic of China.
Ting-Ting
TT
Zhang
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.
Qing-Lin
QL
Meng
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.
Wen-Jun
WJ
Lan
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.
Chun
C
Zhang
CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, People's Republic of China.
Xiao-Mei
XM
Liu
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D051379:Mice; D008813:Mice, Inbred ICR; D060888:Real-Time Polymerase Chain Reaction
D007334:Insulin-Like Growth Factor I
IGF-I; gene doping; gene therapy; internal reference control (IRC); real-time quantitative PCR; recombinant adeno-associated virus (rAAV)