@article{3090537,
    title = "Quadruple-allele dipstick test for simultaneous visual genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) polymorphisms in the toll-like receptor-4 gene",
    author = "Litos, I.K. and Ioannou, P.C. and Christopoulos, T.K. and Tzetis, M. and Kanavakis, E. and Traeger-Synodinos, J.",
    journal = "Clinica Chimica Acta",
    year = "2011",
    volume = "412",
    number = "21-22",
    pages = "1968-1972",
    issn = "0009-8981",
    doi = "10.1016/j.cca.2011.07.001",
    keywords = "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)",
    abstract = "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."
}