双重TaqMan荧光定量PCR在β-地中海贫血产前诊断中的应用

Dual TaqMan Fluorescence Quantitative PCR Applied to Prenatal Diagnosis of beta-Thalassemia

目的建立基因点突变双重TaqMan荧光定量PCR方法,实现β-地中海贫血(β-地贫)高风险胎儿产前基因诊断快速、准确。方法以每反应管检测一个位点的突变和野生型基因序列,建立β-地贫基因点突变双重TaqMan荧光定量PCR体系,检测β-地贫高风险胎儿绒毛标本6例、羊水标本32例,根据荧光PCR阳性扩增结果结合两荧光通道的Ct值差异分析受检标本的基因型,同时以常规RDB检测为对照,分析与评价检测结果。结果 6例胎儿绒毛标本中,RDB检测无法判断结果 1例,经该方法检测为野合纯合子;RDB检测误诊为CD41-42合并CD26双重杂合子1例,经该方法检测为CD41-42杂合子。32例胎儿羊水标本中,RDB检测无法判断的标本1例,经该方法检测为CD41-42杂合子。该3例标本经重新采集胎儿羊水标本复查,验证了定量检测结果的准确性。结论β基因点突变双重TaqMan荧光PCR定量检测方法,可实现β-地贫高风险胎儿的快速准确产前基因诊断,操作简单快速,结果准确可靠,是较为理想的β-地贫产前诊断方法。

Objective To establish a dual Taqman fluorescence quantitative PCR （qPCR） method for point mutation detection, and to realize the fast and accurate prenatal diagnosis of high-risk fetus with beta-thalassemia. Methods A dual Taqman qPCR method for beta-thalassimia point mutation detection was developed according to the design that mutation and wild type sequences of a mutation site analyzed in one reaction tube. This method was also applied to detect 6 villi and 32 amniotic fluid specimens of high-risk fetus with beta-thalassemia. The genotype of a sample was analyzed according to the positive amplification results of qPCR and Ct variance of two fluorescent chan- nels. Meanwhile, reverse dot blot （RDB） method was used as a control for the analysis and evaluation of detection re- suits. Results Of 6 villi specimens, 1 sample which could not be diagnosed with RDB method was detected wild type homozygote by this method,and another sample with the misdiagnosis RDB result of double heterozygote of βCD41-42 and βCD26 was detected heterozygote of βCD41-42 by this method. Of 32 amniotic fluid specimens, 1 sample which could not be diagnosed with RDB method was detected heterozygote of βCD41-42 by this method. All these 3 samples were also re-analyzed and verified with recollected amniotic fluid specimens. Conclusion The fast and accurate pre- natal diagnosis of high-risk fetus with beta-thalassemia can be realized with this dual Taqman qPCR method for beta gene point mutation developed in this study. The method is accurate with easy operation and suitable for prenatal diag- nosis of beta-thalassemia.