用SYBR-GreenⅠ实时荧光PER结合融解曲线分析法诊断α-地中海贫血

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。方法应用SYBR-Greenl进行两个实时荧光聚合酶链反应（real-time fluorescence polymerase chain reaction with SYBR-Green 1，SYBR-PCR），检测左缺（-α^4.2）、右缺（-α^3.7）等位基因，同时进行融解曲线（dissociation calve，DC）和Tm（melting temperature）值分析。PCR产物重组到pCR2．1，重组子梯度稀释作为模板检测灵敏度，确定两种等位基因型（-α^4.2，-α^3.7）的检测下限，并对110份DNA样品进行检测。结果检测-α^4.2和-α^3.7的PCR产物长度分别为1．65kb、1．9kb，Tm分别为（81．5±0．5）℃、（82．5±0．5）℃，检测下限分别为9×10^2个拷贝、4．3×10^2个拷贝。该检测技术的灵敏度较常规PCR结合琼脂糖凝胶电泳法高10倍。结论SYBR-Greenl实时荧光PCR结合融解曲线分析及Tm分析可以灵敏、准确地检测-α^4.2、-α^3.7、αα（包括α^Tα）及--^SEA4种等位基因，从而为各种缺失型α-地中海贫血做出基因诊断。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。

Detection of alpha thalassemia using real-time PCR and dissociation curve analysis]

OBJECTIVE: To establish an automatic, high throughput, quick detection method of alpha thalassemia. METHODS: The genotypes of -alpha(4.2) and -alpha(3.7) were detected by two real-time fluorescence PCRs using SYBR-Green1 (SYBR-PCR) with the analysis of dissociation curve (DC) and melting temperature (Tm). The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique. RESULTS: The length of product of -alpha(4.2) and -alpha(3.7) were 1.65 kb and 1.9 kb respectively, and the Tm were (81.5+/-0.5) degrees C and (82.5+/-0.5) degrees C respectively. The detection deadline were 9 x 10(2 ) copies and 4.3 x 10(2) copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR. CONCLUSION: The genotypes of -alpha(4.2), -alpha(3.7), non-deletion alpha alpha/and --(SEA) can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Green1 combined with the dissociation curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe.