Τίτλος:
Quadruple-allele dipstick test for simultaneous visual genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) polymorphisms in the toll-like receptor-4 gene
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
Background: Toll-like receptor-4 (TLR4) is a central regulators of innate immune response as it interacts with bacterial lipopolysaccharide (LPS) and also with endogenous molecules, such as heat-shock proteins and fibrinogen. Two common single nucleotide polymorphisms, A896G (Asp299Gly) and C1196T (Thr399Ile), have been found in the exon 3 of human TLR4 gene, which lead to structure alteration of the extracellular domain of TLR4 thereby influencing the receptor ability for recognition and ligand binding. Methods: We propose a simple, rapid and reliable method for the simultaneous detection of the two SNPs in TLR4 gene that involves: (a) exponential amplification of the genomic region that spans the two SNPs, (b) quadruple primer extension (PEXT) reaction using two allele-specific primers per SNP, and (c) a simple-to-perform dipstick test that allows visual and simultaneous detection of the four alleles within minutes without the need for specialized instrumentation. Results: The method was applied to the simultaneous detection of the two SNPs in 90 samples of general Greek population and the results showed 100% concordance with those obtained by direct sequencing. The entire assay, starting from genomic DNA, can be run in less than 1.5. h. Conclusions: The dipstick test eliminates multiple incubation and washing steps that are common in microtiter well-based assays and does not require highly trained personnel. Because of these advantages, it is suitable for the routine clinical laboratory or even for point-of-care testing. © 2011 Elsevier B.V.
Συγγραφείς:
Litos, I.K.
Ioannou, P.C.
Christopoulos, T.K.
Tzetis, M.
Kanavakis, E.
Traeger-Synodinos, J.
Περιοδικό:
Clinica Chimica Acta
Λέξεις-κλειδιά:
aspartic acid; genomic DNA; glycine; isoleucine; primer DNA; threonine; toll like receptor 4, allele; article; controlled study; DNA sequence; exon; extracellular space; gene; gene amplification; Greece; human; laboratory test; ligand binding; molecular recognition; priority journal; protein domain; quadruple allele dipstick test; single nucleotide polymorphism; toll like receptor 4 gene, Alleles; G-Quadruplexes; Genotype; Humans; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Toll-Like Receptor 4, Bacteria (microorganisms)
DOI:
10.1016/j.cca.2011.07.001
