XÂY DỰNG QUY TRÌNH KỸ THUẬT REAL-TIME PCR XÁC ĐỊNH BIẾN THỂ RS1801275 TRÊN GEN IL4-RΑ LIÊN QUAN BỆNH VIÊM DA CƠ ĐỊA

Abstract

Objective: Establishing a real-time PCR SYBR procedure to detect rs1801275 variant in the IL4-Rα gene. Methods: pairs of allele-specific oligonucleotide (ASO) primers were designed to detect rs1801275 variant in the IL4-Rα gene. The designed primers were verified their specificity in practical laboratory condition and then were determined their optimal concentration and anealing temperature by conducting real-time PCR reactions with each genotype of the rs1801275. The genotype discriminability of the established procedure was validated through real-time PCR reactions with the rs1801275 recombinant DNA plasmids. Performing the real-time PCR SYBR procedure on 113 samples which the genotype results were confirmed by Sanger sequencing and then evaluating the sensitivity, specificity and accuracy of the real-time PCR procedure by comparing the results from both methods. Results: Successfully developed the SYBR real-time PCR procedure to detect rs1801275 variant in the gene IL4-Rα. ASO primers were specifically designed by Primer-BLAST (NCBI). The optimal concentration of the ASO primers were 250 nM, with the CV% of the Ct values were less than 10% and the average Ct value was 28 ± 2. The |ΔCt| = 3 were the point distinguishes each genotype of rs1801275 (CV% were less than 10%). The sensitivity, specificity, and accuracy of the technique were 100%, 98.9%, and 99.1%, respectively. Conclusion: The real-time PCR SYBR procedure to detect rs1801275 variant in the gene IL4-Rα has been established successfully.