Several human cancers, including cervical cancer, are caused by viral infection. Molecular mechanisms of carcinogenesis generally involve a virally mediated loss of normal cell cycle and growth regulation due to viral integration into the host genome. In cases, viral integration can involve only a few copies to a single copy of viral DNA. In situ polymerase chain reaction (IS-PCR) offers high sensitivity for detecting low copy number viral DNA, but the assay is cumbersome and suffers from reproducibility and artifactual problems. TSA (tyramide signal amplification)-based in situ hybridization offers high sensitivity, but, like IS-PCR, sample throughput is low. This Phase I SBIR proposal aims to develop a sensitive, high throughput method for detecting low copy number viral DNA in cancer cells by combining single cell gel microdrop (GMD) encapsulation technology, in situ hybridization, TSA and flow cytometry. In this method, TSA will be used to amplify the hybridization signals to levels detectable by flow cytometry. In addition to high detection sensitivity and sample throughput, this method will permit detection of rare cancer cells by reducing sample loss through GMD encapsulation. Phase I-research will use cervical cancer as an experimental model.
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