DEVELOPMENT OF A URINE PCR ASSAY FOR HPV DNA DETECTION

人乳头瘤病毒（HPV）是最常见的病毒性传播疾病，高危型HPV与全球90%以上的宫颈癌有关。目前预防宫颈癌的措施包括常规巴氏涂片检查，这似乎是有效的。目前宫颈癌的预防措施包括常规巴氏涂片检查，这似乎是有效的。然而，在美国，一小部分但很重要的女性（>10%）从未做过巴氏涂片检查，或者（>30%）没有常规检查。这可能部分是由于盆腔检查的侵入性和不适。虽然HPV不能在实验室中常规生长，但其DNA可以通过PCR等扩增技术删除。从宫颈拭子或宫颈灌洗液标本中检测HPV DNA已被用作确定HPV感染患病率的流行病学工具。这些程序还需要骨盆检查，限制了其广泛的适用性。一种同样敏感和有效但不需要盆腔检查来检测HPV感染的方法将能够识别更多有宫颈癌风险的女性，并极大地有助于流行病学调查。通过使用扩增技术筛查尿液诊断淋病和衣原体感染的最新进展证明了通过尿液检测诊断宫颈感染的可行性。初步数据已经证明了在HPV感染高危女性的尿液标本中检测HPV DNA的能力。基于这些原因，我们假设尿液PCR检测HPV DNA将反映宫颈感染的状态。本提案的目标是充分开发和验证尿液PCR检测HPV DNA检测，可用于流行病学筛查目的。我们建议通过研究20名尿液中未检测到HPV DNA的妇女，初步建立尿液PCR检测HPV DNA的方法。将测量并优化检测β-珠蛋白DNA（细胞存在的内部对照）和加标至这些尿液标本中的已知量克隆HPV DNA的能力。接下来，将从先前测试的50名妇女中获得尿液，以在宫颈/阴道拭子中检测到HPV DNA。将通过检测这些已知HPV阳性“现场”标本来验证初始实验的提取方法。最后，利用在特定目标#1中优化并在特定目标#2中验证的条件，将招募250名HPV感染高风险女性和250名HPV感染低风险女性的队列。将获得配对尿液和宫颈阴道拭子，并比较检测任何HPV DNA、任何高危HPV DNA和类型特异性HPV DNA的能力。经验证的HPV DNA检测尿液检测可用于更好地定义HPV的流行病学，探索HPV感染的自然史，并识别宫颈癌高危女性。

Human papillomavirus (HPV) is the most common virally sexually transmitted disease, and high-risk types of HPV have been implicated in over 90% of cervical cancers worldwide. Current preventative me measures of cervical cancer include routine Pap smear evaluation, which appears to be effective. Current preventative measures for cervical cancer include routine Pap smear evaluation, which appear to be effective. However, a small but significant proportion (>10%) of females in the United States have either never had a Pap smear or (>30%) do not have them on a routine basis. This may be due, in part, to the invasiveness and discomfort of the requirement pelvic examination. Although HPV cannot be routinely grown in the laboratory, its DNA can be deleted by amplification techniques such as PCR. Detection of HPV DNA from cervical swab or cervical lavage specimens has been used as an epidemiological tool to determine the prevalence rates of HPV infection. These procedures also require a pelvic examination that limits its widespread applicability. A method that is equally sensitive and efficient but does not require a pelvic examination to detect HPV infection will be able to identify more women at risk for cervical cancer and greatly aid in epidemiologic surveys. The recent advances in the diagnosis of gonorrhea and chlamydia infection by screening urine using amplification techniques demonstrate the feasibility of diagnosis a cervical infection by a urine test. Preliminary date have demonstrated the ability to detect HPV DNA in urine specimens from women at high risk for HPV infection. For these reasons, we hypothesize that a urine PCR test for the detection of HPV DNA will reflect the state of infection for the cervic. The goal of this proposal is to fully develop and validate a urine PCR test for HPV DNA detection that can be utilized for epidemiologic screening purposes. We propose to initially develop the urine PCR assay for HPV DNA detection by studying 20 women with no detectable HPV DNA in their urine. The ability to detect beta-globin DNA (internal control for the presence of cells) and known amounts of clon4ed HPV DNA spiked into these urine specimens will be measured and optimized. Next, urine will be obtained from 50 women previously tested to have HPV DNA detected in cervical/vaginal swabs. The extraction method by the initial experiments will be verified by testing these known HPV positive "field" specimens. Finally, utilizing the conditions optimized in specific aims #1 and verified in specific aim #2, a cohort of 250 women at high-risk and 250 women at low-risk for HPV infection will be enrolled. Paired urine and cervico-vaginal swabs will be obtained and the ability to detect any HPV DNA, any high-risk HPV DNA and type-specific HPV DNA will be compared. A validated urine test for HPV DNA detection could be used to better define the epidemiology of HPV, to explore the natural history of HPV infection, and to identify women at higher risk for cervical cancer.