Quadruple-allele dipstick test for simultaneous visual genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) polymorphisms in the toll-like receptor-4 gene

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 1.5h.

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.