Development and validation of a rapid method for genotyping three P‐selectin gene polymorphisms based on high resolution melting analysis
| Autor: | Andrea Čeri, Renata Zadro, Marina Pavić, Margareta Radić Antolic, Ivana Horvat |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Microbiology (medical) Genotyping Techniques genotyping high resolution melting analysis method validation P‐selectin single nucleotide polymorphism Concordance Clinical Biochemistry Single-nucleotide polymorphism Biology Nucleic Acid Denaturation Polymorphism Single Nucleotide High Resolution Melt law.invention 03 medical and health sciences symbols.namesake 0302 clinical medicine law Genotype Humans Immunology and Allergy Genotyping Research Articles Polymerase chain reaction Sanger sequencing Biochemistry (medical) Public Health Environmental and Occupational Health Reproducibility of Results Sequence Analysis DNA Hematology Molecular biology Stroke P-Selectin Medical Laboratory Technology 030104 developmental biology Case-Control Studies 030220 oncology & carcinogenesis symbols |
| Zdroj: | Journal of Clinical Laboratory Analysis. 33:e22698 |
| ISSN: | 1098-2825 0887-8013 |
| Popis: | Background 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, optimize, 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. Methods 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%. Results 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. Conclusion 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. |
| Databáze: | OpenAIRE |
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