Detection of S and Z mutant alleles of SERPINA1 gene by real-time PCR

Abstract

Background: A reliable and fast molecular biological analysis method is needed to detect S and Z mutant alleles of SERPINA1 gene in the practice. In previous studies, conventional polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) and allele-specific PCR methods were used to investigate S and Z mutant alleles of SERPINA1 gene. We designed a real-time PCR (qPCR) method with TaqMan probes to detect S and Z mutant alleles of SERPINA1 gene, and conducted this study to compare with RFLP. Aim: To compared RFLP and real-time PCR (qPCR) methods for detection of SERPINA1 gene mutations and determined the anaytical sensitivity and specificity. Materials and Methods: DNA samples from 473 people were used in the study. M, S, and Z alleles of SERPINA1 gene were detected by qPCR and RFLP. Cases with S and Z mutant alleles were confirmed using Sanger nucleotide sequencing. Kappa coefÏcients, analytical sensitivity and specificity were calculated using MedCalc software for statistical analysis. Results: According to the results, MZ genotype of SERPINA1 gene was detected in 0.21% (n=1), MS genotype in 1.06% (n=5), and SS genotype in 0.42% (n=2). The analytical sensitivity of the RFLP was 71.43% (95% CI, 29.04-96.33%), while the analytical sensitivity of the qPCR was 100.0% (95% CI, 99.21-100.0%). Conclusion: This finding indicating that the qPCR method is more sensitive and suitable compared with RFLP.