摘要
目的建立自动化、高通量、准确快速检测缺失型α地中海贫血基因型的技术。方法运用SYBRGreen1和ABI7000热循环仪分别进行3个实时荧光定量聚合酶链反应(SYBRQPCR),同时进行融解曲线(D.C)和Tm值分析,根据特定Tm值对应的D.C峰值判定基因型,即以该峰值≥Cutoff值定为PCR结果阳性。将PCR产物重组到T载体,筛选到含正确扩增片段的克隆,以梯度稀释重组质粒DNA为模板,进行SYBRQPCR得出标准曲线。从而定量未知标本。结果优化了3个SYBRQPCR反应的引物及其浓度,热循环条件等。检测SEA等位基因的PCR产物长800bp,Tm=82.5℃±1℃。αα等位基因的PCR产物长206bp,Tm=83.0℃±1℃。非SEA等位基因的PCR产物长436bp,Tm=84.0℃±1℃。重组质粒拷贝数在1至105范围内,其对数值与CT值均呈良好线性关系。该检测技术的灵敏度较常规PCR结合琼脂糖凝胶电泳法高出16倍以上。联合运用3个SYBRQPCR反应可以为SEA缺失携带者、缺失型HbH病、非缺失型HbH病、α地贫2纯合子、Bart′s水肿胎儿综合征做出基因诊断,并可用于产前诊断。结论该技术具有自动化程度高、不需荧光标记探针、成本低、易质控、防污染、高通量等优点,适于临床推广应用。
Objective To develop a technique for the detection of genotypes of α-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 α-thalassemia 1 （ αα/--SEA） , Bart＇s Hydrops Syndrom （--SEA/--SEA）, deletion type and non-deletion type of HbH diseases, and homozygote of α-thalassemia-2 were diagnosed by the methods. Conclusion The genotypes of the α-Thalassemia could be diagnosed by the Real-time PCR with SYBR Greenl combined with the melting curve analysis, rapidly, automatically, accurately with high throughput and without dual fluorescent labeled probe.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2005年第8期858-861,共4页
Chinese Journal of Laboratory Medicine
基金
北京市自然科学基金资助项目(JS96004)
