Detection of two exogenous genes in transgenic cattle by loop-mediated isothermal amplification.
Nucleotide-based analytical approaches are indispensable and effective, targeting for the transgenic ingredients in biotechnical products in terms of safety assessment. In this study, a loop-mediated isothermal amplification method was developed for the specific detection of exogenous nucleic acids of hLTF/hLALBA-induced transgenic cattle. The detection limit of the LAMP method was proved to be as low as 10 copies of target molecules in optimized systems, and to be 10-100 times more sensitive than the conventional PCR. Furthermore, fluorescent dye SYBR Green I was used to visualize the color changes of LAMP products by naked eyes in daylight, which resulted in distinct colors between positive and negative reactions. For the detection of transgenes, all the transgenic samples collected from hLTF and hLALBA-induced cattle were amplified by LAMP in 1 h, followed by direct visual SYBR Green I dying or gel electrophoresis. Results showed that transgenic and non-transgenic samples exhibited distinct properties in colors or electrophoresis profiles. Thus, all the results indicated that the LAMP assay was a simple and convenient method for the test of transgenic animals.
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
Shanli
S
Zhai
Chuxin
C
Liu
Qingde
Q
Zhang
Chenyu
C
Tao
Bang
B
Liu
D000818:Animals; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007768:Lactalbumin / Q000032:analysis* / Q000235:genetics; D007781:Lactoferrin / Q000032:analysis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature
D017931:DNA Primers; C490098:LTF protein, human; D007768:Lactalbumin; D007781:Lactoferrin