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Detection of exogenous gene doping of IGF‐I by a real‐time quantitative PCR assay
Author(s) -
Zhang JinJu,
Xu JingFeng,
Shen YongWei,
Ma ShiJiao,
Zhang TingTing,
Meng QingLin,
Lan WenJun,
Zhang Chun,
Liu XiaoMei
Publication year - 2017
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1518
Subject(s) - real time polymerase chain reaction , gene , chemistry , microbiology and biotechnology , polymerase chain reaction , biology , chromatography , biochemistry , computational biology
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.