Nasopharyngeal carcinoma (NPC) is a squamous cancer of the head and neck which affects the nasopharyngeal epithelium. Since NPC cells express Epstein-Barr Viral (EBV) genes, a double-stranded DNA herpes virus, its tumours release circulating tumour DNA (ctDNA) containing EBV into circulation. Despite having uses for detection, staging, and screening, plasma EBV DNA analysis is particularly useful for post-treatment prognostication, or assessment of disease course. Due to the positive relationship between plasma EBV DNA concentration and tumour volume, EBV DNA clearance after radiotherapy is predictive of treatment outcome. Detecting small amounts of DNA is vital. The purpose of this project is to assess the current practice of using quantitative polymerase chain reaction (qPCR) or droplet digital PCR (ddPCR) to interrogate a specific gene associated with EBV, as this has a false positive rate of 7%. ddPCR, which measures absolute nucleic acid quantity by counting molecules encapsulated in discrete, volumetrically defined partitions, is hypothesized to have a similar detection limit as qPCR. First, genomic DNA (gDNA) from an EBV+ cell line and EBVcell line are isolated, purified, and sheared. Then, variable amounts of EBV DNA are detected using either qPCR or ddPCR. The results show that the size selected EBV+ and EBV- gDNA are approximately 200 base pairs, and that qPCR and ddPCR have similar detection limits for the EBV genome. In conclusion, more sensitive DNA quantification techniques than qPCR and ddPCR are needed for clinical use.
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