Τίτλος:
Lateral flow dipstick test for genotyping of 15 beta-globin gene (HBB) mutations with naked-eye detection
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (. HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896. bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ~2. h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood. © 2012 Elsevier B.V.
Συγγραφείς:
Papanikos, F.
Iliadi, A.
Petropoulou, M.
Ioannou, P.C.
Christopoulos, T.K.
Kanavakis, E.
Traeger-Synodinos, J.
Περιοδικό:
Analytica Chimica Acta
Λέξεις-κλειδιά:
Dipstick; Genotyping; Hemoglobinopathies; Lateral Flow; Thalassemia; Visual detection, Diagnosis; Eye protection; Polymerase chain reaction, Genes, beta globin; biotin, article; DNA determination; gene; gene mutation; genotype; genotyping technique; HBB gene; lateral flow dipstick test; nucleotide sequence; observational method; polymerase chain reaction; priority journal, beta-Thalassemia; DNA Mutational Analysis; Genotype; Globins; Humans; Mutation; Point-of-Care Systems; Polymerase Chain Reaction
DOI:
10.1016/j.aca.2012.03.041
