Sensitive Detection of PIK3CA Exon 20 H1047R Breast Cancer based on Low-Cost Intercalary Dye SYBR Green I Real-Time qPCR Assay

• DOI: 10.17576/jsm-2024-5311-12
• Publication Date: 2024-11-28T04:36:24Z
• Authors: Desriani Desriani; Azamris Azamris; Primariadewi Rust...; Inherni Marti Abna; Irsyad Ibadurrahman; Asrul Muhammad Fuad; Dini Nurdiani; Yuliawati Yuliawati; Nuruliawaty Utami; Neng Herawati; Najmiatul Fitria; Muhammad Ali Wari...
• Keywords: SYBR Green I

Breast cancer is associated with an excessive function of somatic mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), specifically exon 9 and 20 hotspots. Exon 20, which contains H1047R, has more potential for oncogenic than 9. Detection PIK3CA/H1047R also been reported molecular diagnosis therapeutic application. Sensitive methods are required because mutants mostly present at low levels a mixture the wild type. Therefore, this study aimed to explore development low-cost sensitive PIK3CA H1047R detection method using intercalary dye real-time qPCR (quantitative polymerase chain reaction), SYBR Green I. The was primer design, formulation, specificity, limit detection, reproducibility, repeatability, genotyping 15 DNA (deoxyribonucleic acid)-frozen tissue patient samples, were further confirmed by PCR sequencing validation. proportion formulations 0.25 µM (WT), 0.65 H1047 (MT), 0.2 reverse 10 μl total volume. results showed that compared gold standard (6.6-20%), developed had higher sensitivity 5%. coefficients intra- inter-variability between 0.01-0.19%, suggesting repeatable reproducible mean pipetting error. Genotyping breast samples wild-type genotype, 100% agreement result. sensitive, reproducible, results, potentially applicable prognosis predictions.