engleski

Development and validation of a rapid method for genotyping three P‐selectin gene polymorphisms based on high resolution melting analysis

High resolution melting (HRM) analysis is one of the newer, reliable and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P- selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimise and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T) and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion ≥95%. Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. The validation confirmed PCR-HRM as a precise, accurate and specific method for genotyping the c.992G>A, c.1918G>T and c.2266A>C SELP polymorphisms.

genotyping, high resolution melting analysis, method validation, P‐selectin, single nucleotide polymorphism

Rad/časopis indeksiran je i u: Natural Science Collection ; Nutrition Abstracts & Reviews Series A ; ProQuest Central ; Public Health Database ; PubMed Dietary Supplement Subset ; SciTech Premium Collection.