Comparison between probe melting curve analysis based on real-time fluorescent PCR and reverse dot blot assay for gene and prenatal diagnosis of β-thalassemia

Abstract

Objective To evaluate the reliability of the probe melting curve analysis (PMCA) based on real-time fluorescent PCR assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia. Methods A total of 135 cases of peripheral blood samples were collected from Zhuhai Municipal Maternity and Child Healthcare Hospital between 2008 and 2010.All the samples were performed preliminary diagnosis according to the hematological data.Of these, 119 cases were diagnosed as β-thalassemia trait, 4 cases were diagnosed as severe thalassemia and 12 cases were normal.In addition, 44 cases of amniotic fluid and 8 cases of cord blood with high-risk for severe β-thalassemia were also collected.The diagnostic reliability of the PMCA assay and reverse dot blot assay was evaluated on 187 above-mentioned cases by direct DNA sequencing analysis in a double-blind study. Results The genotypes of 185 cases were detected accurately based on the PMCA assay except for two cases:one heterozygote with Ini(ATG>AGG) was omitted and another heterozygote couldn′t be distinguished between CD43(G>T) and CD37(G>A).For the RDB assay, only one heterozygote with CD71-72(+T) was not detected accurately in the above-mentioned cases.Compared with the DNA sequencing analysis, the sensitivity, specificity, negative predictive value, positive predictive value and diagnostic efficiency of the PMCA assay were 98.75%, 100.00%, 93.10%, 100.00% and 98.93%, respectively.The corresponding value of the RDB assay were 99.38%, 100.00%, 96.42%, 100.00% and 99.47%, respectively.There were no significant between-group differences in the diagnostic efficiency of the two assays (P>0.05).The results of prenatal diagnosis were in complete concordance with the follow up results, after the birth or induced labour of the fetuses. Conclusions The PMCA assay can be used as an alternative and verified method of RDB assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia.(Chin J Lab Med, 2012，35：413-417) Key words: beta-Thalassemia; Polymerase chain reaction; Melting curve analysis; Nucleic acid hybridization; Prenatal diagnosis