DISCRETE ASSAY FOR INTEGRATED HPV DNA IN CERVICAL CELLS

目前的研究表明，宫颈癌的发生与人类乳头瘤病毒(HPV)几个亚型的DNA整合密切相关。巴氏试验有助于早期发现前驱病变；然而，当检测到低级别病变时，结果并不确定。在这些病例中，基于溶液的杂交方法可用于筛查与宫颈癌相关的常见HPV亚型的存在和病毒载量。然而，HPV的存在并不是宫颈癌后续发展的有用指标。可以确定病毒整合发生的检测是疾病的更好指标。目前的原位杂交方法在区分整合体病毒和异体病毒方面的成功有限。分析很困难，因为异型病毒和整合型病毒通常并存，而表明异型病毒的“弥漫”模式往往掩盖了表明整合型病毒的“点”模式。通过结合单细胞凝胶微滴(GMD)包埋技术、原位杂交和核分裂技术，这一第一阶段的建议旨在开发一种明确区分整合病毒和异体病毒的方法。GMD技术的使用还将减少细胞损失，便于检测稀有细胞。拟议的商业应用：该分析将使宫颈癌的早期检测成为可能，并对与宫颈癌直接相关的病毒致病机制进行高度特异性的评估，从而促进早期治疗并降低死亡率。

Current studies indicate that development of cervical cancer is strongly linked to the DNA integration of several subtypes of the human papillomavirus (HPV). The Pap test has helped in the early detection of precursor lesions; however, results are inconclusive when low grade lesions are detected. In these cases, a solution based hybridization method can be used to screen for the presence and viral burden of common HPV subtypes associated with cervical cancer. The presence of HPV, however, is not a useful indicator of subsequent development of cervical cancer. A test which can determine the occurrence of viral integration is a better indicator of disease. Current in situ hybridization methods have limited success in distinguishing integrated virus from episomal virus. Analysis is difficult because both episomal and integrated virus often coexist, and the "diffuse" pattern indicating episomal virus often masks the "dot" pattern indicating integrated virus. By combining single cell gel microdrop (GMD) encapsulation technology, in situ hybridization and nuclear fractionation techniques, this Phase I proposal aims to develop an assay which will unambiguously distinguish integrated virus from episomal virus. Use of GMD technology will also reduce cell loss, facilitating rare cell detection. PROPOSED COMMERCIAL APPLICATIONS: The assay will permit early detection of cervical cancer and highly specific assessment of viral pathogenic mechanisms directly linked to cervical cancer, facilitating early treatment and reducing mortality rates.