Περίληψη:
Background: Hemoglobinopathies are the most common inherited diseases worldwide. Various methods for genotyping of hemoglobin, beta (HBB) gene mutations have been reported, but there is need for a high sample-throughput, cost-effective method for simultaneous screening of several mutations. We report a method that combines the high detectability and dynamic range of chemiluminescence with the high allele-discrimination ability of probe extension reactions for simultaneous genotyping of 15 HBB mutations in a high sample-throughput, dry-reagent format. Methods: We genotyped the HBB mutations IVSI-110G>A, CD39C>T, IVSI-1G>A, IVSI-6T>C, IVSII-745C>G, IVSII-1G>A, FSC6GAG>G-G, -101C>T, FSC5CCT>C-, IVSI-5G>A, FSC8AAG>-G, -87C>G, IVSII-848C>A, term+6C>G, and HbS (cd6GAG> GTG). The method used comprises the following: (a) duplex PCR that produces fragments encompassing all 15 mutations, (b) probe extension reactions in the presence of fluorescein-modified dCTP, using unpurified amplicons, and (c) microliter well-based assay of extension products with a peroxidase- antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents to simplify the procedure and assigned the genotype by the signal ratio of the normal-to-mutant-specific probe. Results: We standardized the method by analyzing 60 samples with known genotypes and then validated by blindly genotyping 115 samples with 45 genotypes. The results were fully concordant with sequencing. The reproducibility (including PCR, probe extension reaction, and chemiluminometric assay) was studied for 20 days, and the CVs were 11%-19%. Conclusions: This method is accurate, reproducible, and cost-effective in terms of equipment and reagents. The application of the method is simple, rapid, and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations. © 2007 American Association for Clinical Chemistry.
Συγγραφείς:
Glynou, K.
Kastanis, P.
Boukouvala, S.
Tsaoussis, V.
Ioannou, P.C.
Christopoulos, T.K.
Traeger-Synodinos, J.
Kanavakis, E.
Λέξεις-κλειδιά:
fluorescein derivative; hemoglobin beta chain; horseradish peroxidase; peroxidase, allele; amplicon; article; beta thalassemia; chemoluminescence; controlled study; gene amplification; gene mutation; genotype; high throughput screening; human; major clinical study; microtiter plate assay; polymerase chain reaction, beta-Thalassemia; Chemiluminescent Measurements; Genetics, Population; Genotype; Globins; Humans; Mutation; Polymerase Chain Reaction; Reagent Kits, Diagnostic; Reproducibility of Results
