QUY TRÌNH KỸ THUẬT REAL-TIME PCR SYBR CHẨN ĐOÁN BIẾN THỂ RS216321 TRÊN GEN VWF Ở NGƯỜI BỆNH XUẤT HUYẾT NỘI SỌ TỰ PHÁT

Abstract

Objective: To develop a SYBR Green real-time PCR assay to detect the rs216321 variant gene VWF in patients with spontaneous intracranial hemorrhage. Materials and methods: Designing three allele-oligonucleotide primers, evaluating their specificity and optimizing their concentration. Determining the diagnostic characteristics of the rs216321 variant by performing a real-time PCR reaction on the QuantStudio 5 system (Thermo Fisher). Applied the optimized procedure to 22 Sanger-sequenced samples to evaluate sensitivity, specificity and accuracy. Results: Successfully built real-time PCR process, including designing and evaluating the specificity of three primers using the Labchip gel electrophoresis method (with one clearly separated light intensity peak), optimizing the primer concentration (250 nM) and determining the absolute value of ΔCt to be 3 in order to distinguish genotypes. The sensitivity, specificity and accuracy reached 100 percent. Conclusions: Built a real-time PCR to identify the single nucleotide polymorphic variant rs216321 in VWF gene. This procedure was applied to patients, providing a basis for surveying a large, representative sample size. This creates a source of data and information about variations in the Vietnamese population.