实时荧光PCR熔解曲线法在β-地中海贫血基因诊断和产前诊断中的临床应用评价

The evaluation of a melting curve analysis-based PCR assay for the clinical genetic diagnosis and prenatal diagnosis of β-thalassemia

目的对荧光PCR熔解曲线法用于β-地中海贫血（β-地贫）基因诊断和产前诊断进行临床评价。方法收集2011年1至8月柳州市妇幼保健院外周血标本451份，其中经血液学表型分析为β-地贫阳性表型标本372份，β-地贫阴性表型标本79份。同时收集2011年6至9月夫妇双方经PCR-RDB探针法确诊为B一地贫基因携带者，在我院行β-地贫产前诊断的胎儿绒毛、羊水及脐带血标本共84份，其中孕10～13周胎儿绒毛标本16份、孕16～24周羊水标本64份、孕17周以上胎儿脐血标本4份。按双盲对照试验，分别采用荧光PCR熔解曲线法（可检测24种13珠蛋白基因突变）、PCR-反向点杂交（RDB）探针法（可检测17种13珠蛋白基因突变）和DNA测序法同时对451份外周血标本和84份胎儿绒毛、羊水、脐带血产前诊断标本进行检测。计算荧光PCR熔解曲线法和PCR—RDB探针法、DNA基因测序法检测结果的野生型符合率、突变型符合率和总符合率。结果利用荧光熔解曲线法在451份外周血标本中共检出13种突变类型、19种基因型，其中有447份标本与PCR．RDB探针法的基因型检测结果相符，符合率为99．1％（447／451），902个等位基因位点检出符合率为99．6％（898／902），有4份标本与PCR—RDB探针法的基因型检测结果不相符，经DNA测序法验证，有3份标本与荧光熔解曲线法的检测结果完全一致，1份标本的13珠蛋白基因突变不在荧光熔解曲线法检测范围而未被检出。450份外周血标本的荧光熔解曲线法检测结果与DNA测序法相符，符合率为99．8％（450／451）。利用荧光熔解曲线法在84份产前诊断胎儿标本中共检出8种突变类型、18种基因型，168个等位基因位点的检测结果与PCR—RDB探针法和DNA基因测序法检测结果符合率为100％。结论荧光PCR熔解曲线法可同时检测多份待测标本，并可准确检出多种基因突变类型，可用于β-地贫的基因诊断和产前诊断。

Objectives To investigate the clinical value of the melting curve analysis-based PCR assay for the clinical genetic diagnosis and prenatal diagnosis of β-thalassemia. Methods A total of 451 peripheral blood samples, including 372 cases with β-thalassemia phenotypes and 79 eases without β-thalassemia phenotypes, were collected by Liuzhou Municipal Maternity and Child Healthcare Hospital between January 2011 and August 2011. Moreover, another 84 eases, including 16 fetal villi samples （10-13 weeks）, 64 amniotic fluid samples （16 -24 weeks） and 4 umbilical cord blood samples （above 17 weeks）, whose parents were β-thalassemia carriers, were also collected for this assay between June 2011 and September 2011. A double-blind test was done to compare the detection reliability of the melting curve analysis-based assay （24 β-thalassemia mutations can be detected） with PCR-RDB probe assay （17 β-thalassemia mutations can be detected） and DNA sequencing using these samples. The wild- type, mutant and total concordance rates of the genotyping results were calculated separately among the melting curve analysis based assay, PCR-RDB probe assay and DNA sequencing. Results Among the 451 peripheral blood samples, thirteen mutations and nineteen genotypes were obtained by using melting curve analysis-based assay. 447 samples had the same detection results and 4 samples had different detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay, thus, the concordance rate of the sample detection result was 99. 1% （447/451） , and the concordance rate of the allele detection result was 99. 6% （ 898/902 ）. DNA sequencing results of the 4 samples showed that 3 samples had the same genotyping result with melting curve analysis-based assay, and 1 sample had the same genotyping result with PCR-RDB probe assay. A rare β-globin mutation which was not included by melting curve analysis-based assay was not detected. Thus, the genotypes of 450 samples were detected accurately by melting curve analysis-based assay, and the concordance rate of the sample detection between the melting curve assay and DNA sequencing assay was 99.8% （450/451）. Among 84 fetal villi, amniotic fluid, and umbilical cord blood samples, 8 mutation types and 18 genotypes were obtained by using melting curve analysis-based assay. All the samples have the same detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay and DNA sequencing, so the concordance rate of the genotyping results was 100% among the melting curve analysis-based assay, PCR-RDB probe assay and DNA sequencing. Conclusions The melting curve analysis-based PCR assay can detect multiple unknown samples simultaneously, and detect multiple mutations accurately. It is very useful for the genetic diagnosis and prenatal diagnosis of β- thalassemia.