PCR-荧光探针法在缺失型α-地中海贫血基因诊断中的应用

Application of PCR-fluorescence probe method in genetic diagnosis of deletional α-thalassemia

目的：探讨PCR-荧光探针法在缺失型α-地中海贫血基因诊断中应用的可行性。方法：选取2014年4-6月在该实验室检测的303例样本,经基因DNA提取后,按双盲对照试验,分别采用缺口PCR（Gap-PCR）和PCR-荧光探针法对各样本进行α-地中海贫血基因缺失突变检测,比较两种方法基因诊断的符合率,对两种技术检测结果不吻合的样本采取多重连接探针扩增（MLPA）技术进行验证。结果：应用Gap-PCR技术检测出α-珠蛋白基因缺失突变175例,无α-珠蛋白基因缺失突变128例;PCR-荧光探针法检测出α-地中海贫血基因缺失样本173例,无α-地中海贫血基因缺失样本130例,其中有2个样本与Gap-PCR方法的结果不符合,经MLPA方法验证2个样本结果与Gap-PCR结果一致。与Gap-PCR方法相比,PCR-荧光探针法对α-地中海贫血基因缺失检测的阳性符合率为98.85%（173/175）、阴性符合率为100.00%（128/128）、Kappa值为0.987,总符合率为99.34%（301/303）。结论：PCR-荧光探针法用于缺失型α-地中海贫血的检测,具有快速、准确、简单实用、高通量低成本等优点,可结合血液学初筛技术应用于缺失型α-地中海贫血基因诊断。

Objective： To explore the feasibility of PCR-fluorescence probe method in genetic diagnosis of deletional α-thalassemia. Methods： A total of 303 specimens detected in the laboratory from April to June in 2014 were selected, after：abstracting gene DNA, Gap-PCR and PCR-fluorescence probe method ~ere used to detect deletion mutation of α-thalassemia gene respectively by a double-blind controlled study, the coincidences rate of the two methods for genetic diagnosis were compared, the inconsistent specimens of the two methods were verified by muhiplex ligation-dependent probe amplification （MLPA） . Results： A total of 175 cases with deletion mutation of α-globin were found by Gap-PCR, and 128 cases had no deletion mutation of α-globin; 173 cases with ct-thalassemia gene deletion were found by PCR-fluorescence probe method, and 130 cases had no α-thalassemia gene deletion, the results of two specimens were not meet the results of Gap-PCR, after venfication by MLPA, the results of the two methods in the two specimens were consistent. Compared with Gap- PCR, the positive concordance rate, negative concordance rate, Kappa value, and the total concordance rate of PCR-fluorescence probe method in genetic diagnosis of deletional α-thalassemia were 98.85% （173/175） , 100. 00% （128/128）, 0. 987, and 99.34% （301/ 303）, respectively. Conclusion： PCR-fluorescence probe method used for detection of deletional α-thalassemia has the advantages of speediness, accuracy, simpleness, practicality, high throughput, and low cost, which can be used for genetic diagnosis of deletional α- thalassemia combining with hematologic primary screening technology.