DEVELOPMENT OF A URINE PCR ASSAY FOR HPV DNA DETECTION

人乳头瘤病毒（HPV）是最常见的病毒性性传播疾病，全球90%以上的宫颈癌与高危型HPV有关。目前宫颈癌的预防措施包括常规子宫颈抹片检查，这似乎是有效的。目前宫颈癌的预防措施包括常规子宫颈抹片检查，这似乎是有效的。然而，美国有一小部分（10%）女性从未做过子宫颈抹片检查，或者（30%）没有做常规检查。这可能部分是由于盆腔检查的侵入性和不适。虽然人乳头瘤病毒不能在实验室中常规生长，但它的DNA可以通过PCR等扩增技术删除。从宫颈拭子或宫颈灌洗标本中检测HPV DNA已被用作确定HPV感染流行率的流行病学工具。这些手术还需要盆腔检查，这限制了其广泛的适用性。一种同样灵敏和有效，但不需要盆腔检查来检测HPV感染的方法将能够识别出更多有宫颈癌风险的妇女，并极大地有助于流行病学调查。最近的进展，诊断淋病和衣原体感染筛查尿扩增技术证明了可行性诊断宫颈感染的尿液测试。初步数据证明了在HPV感染高危妇女尿液标本中检测HPV DNA的能力。由于这些原因，我们假设尿液PCR检测HPV DNA将反映宫颈感染的状态。本提案的目的是充分开发和验证尿PCR检测HPV DNA，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，初步开发尿液PCR检测HPV DNA的方法。检测-珠蛋白DNA（细胞存在的内部控制）和已知数量的克隆HPV DNA加入这些尿液样本的能力将被测量和优化。接下来，将采集50名女性的尿液，这些女性之前曾在宫颈/阴道拭子中检测过HPV DNA。初步实验的提取方法将通过检测这些已知的HPV阳性“现场”标本来验证。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感染高危妇女和250名低风险妇女。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和特定类型HPV DNA的能力。有效的尿检HPV DNA检测可用于更好地定义HPV流行病学，探索HPV感染的自然史，并确定宫颈癌高危妇女。

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.