Article Sidebar
Views PDF: 156
OPTIMIZED ALLELE-SPECIFIC REAL-TIME PCR ASSAYS FOR THE DETECTION OF THE rs216321 VARIANT IN VWF GENE IN PATIENTS WITH SPONTANEOUS INTRACRANIAL HEMORRHAGE
Main Article Content
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.
Article Details
References
2. Scotland G, Biobank MGB, Biobank U, et al. Global Biobank Meta-analysis Initiative: Powering genetic discovery across human disease. Cell Genomics. 2022:9.
3. Alzahrani FM, Aldossary N and Hassan FM. Phenotypic and Genotypic Characterization of von Willebrand Factor Gene (Exon 18 and 20) in Saudi Healthy Individuals. Med Arch. 7. 2020; 74 (5):337-341. 10.5455/medarh.2020.74.337-341.
4. Elek Z, Losoncz E, Maricza K, et al. Missense Variants of von Willebrand Factor in the Background of COVID-19 Associated Coagulopathy. Genes (Basel). 2023; 14(3). 10.3390/genes14030617.
5. Guo H, You M, Wu J, et al. Genetics of spontaneous intracerebral hemorrhage: risk and outcome. Front Neurosci. 2022; 16:455.
6. Han J-H, Lee J-M, Koh E-J and Choi H-YJJOKNS. The spot sign predicts hematoma expansion, outcome, and mortality in patients with primary intracerebral hemorrhage. J Korean Neurosurg Soc. 2014; 56(4):303-309.
7. Hemphill Iii JC, Greenberg SM, Anderson CS, et al. Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2015; 46(7):2032-2060.
8. National Library of Medicine (NIH). Primer-BLAST. October 5th, 2023. October 5th, 2023. https://www.ncbi.nlm.nih.gov/tools/primer-blast/.
9. ThermoFisher Scientific. Precision in qPCR. October 5th, 2023. October 5th, 2023. https://www.thermofisher.com/vn/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/gene-expression-analysis-real-time-pcr-information/precision-qpcr.html.