Document Type

Theses, Ph.D

Disciplines

1.4 CHEMICAL SCIENCES, 1.6 BIOLOGICAL SCIENCES

Publication Details

A Report Presented for the award of PhD. Research Carried Out Under the Guidance of Dr. Luke McGuigan and Dr. Denise Cornally Submitted to Technological University Dublin September 2025.

Abstract

Human Papillomaviruses (HPV) belong to the Papillomaviridae family, with over 200 known types classified into high-risk (HR-HPV) and low-risk (LR-HPV) groups based on their carcinogenic potential. While most HPV infections are asymptomatic and transient, persistent infection can lead to health issues, including cervical cancer. Given the high prevalence of HPV and its potential for malignant transformation, nucleic acid amplification assays have become a widely implemented tool for routine HPV testing. This study developed a real-time isothermal DNA amplification assay for the detection and differentiation of multiple HPV types. A universal primer set was designed to target a highly conserved region of the HPV L1 gene, generating a 107–110 bp amplicon. Concurrently, molecular beacon probes were developed and assessed for HPV 16, HPV 18, HPV 31, and HPV 45 to allow for real-time detection and discrimination. Initial primer assessment using a SYBR Green-based PCR assay confirmed the robustness and specificity of the universal primer set. Melt curve analysis, agarose gel electrophoresis, and Sanger sequencing further verified the designed universal primers' specificity across multiple HPV types. The incorporation of molecular beacon probes exhibited amplification plots with high sensitivity, low variation, and strong linearity (R² > 0.99) for the HPV types under PCR analysis. Critical reaction components and conditions were optimised for isothermal amplification to enhance assay sensitivity and specificity. The isothermal reaction time and incubation temperature were evaluated to identify optimal reaction conditions. The concentration of reagents, including Bst DNA Polymerase, ATP, and Tte UvrD helicase, was altered to optimise assay specificity and reproducibility. The molecular beacon concentration was evaluated with ii a final concentration of 0.15 µM, providing the best signal-to-noise ratio and consistent amplification across replicates. Additionally, an asymmetrical primer ratio was determined to enhance amplification efficiency under isothermal conditions. The inclusion of the oligopeptide antibiotic Netropsin demonstrated its ability to reduce non-specific amplification. However, Netropsin was found to also inhibit target amplification and detection. The omission of Netropsin did not negatively impact the performance of the HPV ISOBeacon assay. This study successfully established a real-time HPV ISOBeacon assay that combines a universal primer set capable of detecting four HR-HPV types under isothermal amplification conditions. Additionally, the incorporation of four molecular beacon probes for the detection and discrimination of four HR-HPV types. The developed HPV ISOBeacon assay demonstrated reproducible performance, suggesting it to be a promising addition to HPV diagnostics, supporting the WHO’s call for expanded assay availability to expand cervical cancer screening programmes.

Creative Commons License

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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