[Rapid diagnosis of Down's syndrome by multiplex real-time fluorescence relative quantitative PCR].
To establish a multiplex real-time fluorescence relative quantitative PCR method for diagnosis of Down's syndrome. The fragment from Down's syndrome critical region gene 3 (DSCR3) on chromosome 21 was used as the target gene, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene on chromosome 12 was used as the control gene. The two genes were amplified in the same tube. The relative quantitative index-CT value was used to differentiate trisomy 21 patient from normal person. The peripheral blood sample from a Down's syndrome patient was collected and the B-lymphocytes were transformed by Epstein-Barr virus to establish the immortalized cell lines as standard material. The reaction conditions were optimized to obtain an equal amplification efficiency from both the target and the control genes. The slopes of both genes were almost -3.32, indicating that the efficiencies of the two amplifications were approximately equal. Among a certain range from 3-300 ng/PCR, the variation of detected DeltaCT value were less than 15%, and amplification showed the highest reproducibility when the concentration of DNA template was 30 ng/microL. Then, the variation of DeltaCT value with inter- and intra-assay were 9.8% and 13.3% at this DNA concentration of the templates. Clinical samples, including 20 blood samples from patients and 30 blood samples from normal persons, were detected using the established method. The DeltaCT value from Down's syndrome group were dramatically different from normal group (P < 0.001). The trisomy 21 immortalized cell lines were established and the genetic integrity of the cell lines was stable as evaluated by karyotype and DNA analysis. The relative quantitative PCR with DeltaCT value could be used to rapidly diagnose Down's syndrome.
Basic Medical Research Center, Capital Medical University Affiliated Beijing Chaoyang Hospital, Beijing 100020, China. wenwugao@sina.com
Bin
B
Gao
Bai
B
Xiao
Qi-Lian
QL
Zou
Shang-Zhi
SZ
Huang
Li-Rong
LR
Wang
Shu-Lan
SL
Niu
Mei
M
Yan
Zhen
Z
Lei
Xing-Yuan
XY
Jia
Zhan-Yong
ZY
Wang
Hai-Xin
HX
Yuan
Yan
Y
Wu
Jing-Zhong
JZ
Liu
D002460:Cell Line; D004314:Down Syndrome / Q000175:diagnosis* / Q000235:genetics*; D005453:Fluorescence; D006801:Humans; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods*