Development and validation of a quantitative pcr array assay for the detection of viral sequences in breast cancer

• A qPCR Multiplex Array assay for simultaneous detection of 21 viral sequences was developed. • The assay demonstrated high specificity, linearity (R 2 > 0.98), and reproducibility (CV < 10%). • Amplification efficiencies within the 90–110% range confirmed reliable quantitative performance. • HPV, MMTV, and EBV sequences were detected in 30%, 24%, and 0.6% of breast cancer cases. • Sanger sequencing confirmed 100% sequence identity in all qPCR-positive samples. To develop and validate a quantitative PCR Multiplex Array assay for the detection of viral sequences in breast cancer (BC) tissue, and to assess its potential utility as a molecular research and diagnostic tool. A total of 172 formalin-fixed paraffin-embedded (FFPE) biopsy samples from patients with a histological diagnosis of BC, and 10 non-neoplastic breast tissue samples were collected at the High Specialty Regional Hospital “Ciudad Salud” (HRAECS). The study was approved by the Institutional Research and Ethics Committees. Samples were analyzed using a qPCR Multiplex Array assay. gBlocks Gene Fragments at 10 ng/µL were used as positive controls for each target virus. Assay validation demonstrated high specificity, linearity (R 2 > 0.98), amplification efficiencies within the 90–110% range, and reproducibility (CV < 5%), confirming the reliability of this method for viral detection in clinical tissue samples. Viral sequences were detected in 30% (51/172) of cases for HPV, 24% (41/172) for MMTV, and 0.6% (1/172) for EBV. Sanger sequencing confirmed 100% sequence identity with GenBank reference sequences in all positive samples. A qPCR Multiplex Array assay targeting 21 viral sequences was developed and validated, providing a reliable tool for the detection of cancer-associated viral sequences in BC tissue. Detection of viral sequences confirms their presence in tumor tissue but does not establish causality with carcinogenesis. Future studies integrating transcriptomic, functional, and clinical analyses are needed to clarify the role of these viruses in BC, including associations with clinicopathological features and potential therapeutic implications.