Detection of ΔF508del using quantitative real-time PCR, comparison of the results obtained by fluorescent PCR

Objective: Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in the Caucasian population. The molecular diagnosis is difficult since there are about 1,000 mutations in the CF transmembrane regulator gene. The Delta F508del is the cause of the CF in 64% of the cases in Hungary. Our aim was to compare two polymerase chain reaction (PCR)-based method for the detection of Delta F508del. Methods: One hundred and sixteen DNA samples isolated from different tissues (84 blood samples, 18 chorionic villus samples and 14 amniotic fluid samples) were involved in the study. Fluorescent PCR with DNA fragment analysis and quantitative real-time PCR with melting curve analysis were performed on the DNA samples for the detection of Delta F508del. Results: Sixty-five healthy normal samples, 43 heterozygous samples, 6 Delta F508del homozygous samples and 2 Delta F508C homozygous samples were detected by using quantitative real-time PCR combined with melting curve analysis. The fluorescent PCR method did not detect the Delta F508C mutation and these two samples were diagnosed as normal healthy ones. Conclusions: The quantitative real-time PCR with melting curve analysis is a reliable and fast method for the detection of Delta F508del. The results are ready in 1 h following the DNA isolation. The applied primer-probe set with melting curve analysis gives additional information for the presence of other mutations in the Delta F508del region. Copyright (c) 2007 S. Karger AG, Basel.