Molecular diagnosis of ?-thalassemia by using the real time PCR combined with the dissociation curve analysis
- VernacularTitle:实时荧光聚合酶链反应结合融解曲线分析在?-地中海贫血基因诊断中的价值
- Author:
Jingzhong LIU
;
Mei YAN
;
Lirong WANG
;
Zhanyong WANG
;
Yan ZHOU
;
Bai XIAO
- Publication Type:Journal Article
- Keywords:
Real-time PCR;
alpha-Thalassemia
- From:
Chinese Journal of Laboratory Medicine
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a technique for the detection of genotypes of ?-thalassemia, which was rapid and automatical with high through-put.Methods The Real Time PCR and dissociation curve analysis (D.C) were carried out by using SYBR Green1 on ABI7000.Positive results of the 3 SYBR-Q-PCRs were defined by the -dF/t℃≥cut off value of the peak at the specific Tm for each right PCR product.Three PCR products were recombined with the T-vector.The correct positive clones were selected by sequencing.Standard curves of the CT vs. log values of copies were done from a serious dilutions of the recombinant plasmid DNA which served as the template in SYBR-Q-PCRs.Results The conditions of the PCR including concentrations of primers, thurmocycler program etc.were optimized.Specific Tm values for the 3 SYBR-Q-PCRs were 82.5?1℃, 83.0?℃ and 84.0?1℃ respectively. The standard curves for p800 bp and p206 bp DNA showed a good linear relationship between CT and log value of the copies of the template DNA ranging from 10~5 copies to single copy. Sensitivity of the technique were at least 10~1 to 10~2 times higher than that of the regular PCR plus gel electrophoresis method.The techniques and reagents showed very good reproducibility and stability besides the high sensitivity. The ?-thalassemia 1(??/--SEA), Bart′s Hydrops Syndrom (--SEA/--SEA), deletion type and non-deletion type of HbH diseases, and homozygote of ?-thalassemia-2 were diagnosed by the methods.Conclusion The genotypes of the ?-Thalassemia could be diagnosed by the Real-time PCR with SYBR Green1 combined with the melting curve analysis.rapidly, automatically, accurately with high throughput and without dual fluorescent labeled probe.
