ESTABLISHING A REAL-TIME PCR SYBR PROCEDURE FOR DETECTING rs1801275 VARIANT IN THE IL4-Rα GENE ASSOCIATED WITH THE ATOPIC DERMATITIS

Thị Thôi Lê, Quốc Đạt Ngô , Minh Hà Nguyễn, Hữu Ngọc Tuấn Nguyễn

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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.

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References

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