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

Abstract

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 cases without β-thalassemia phenotypes, were collected by Liuzhou Municipal Maternity and Child Healthcare Hospital between January 2011 and August 2011.Moreover, another 84 cases, 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.(Chin J Lab Med, 2012，35：407-412) Key words: beta-Thalassemia; Polymerase chain reaction; Melting curve analysis; Genetic diagnosis; Prenatal diagnosis