HPV DIAGNOSIS BY PCR IN CERVICAL CANCER STUDIES

宫颈癌研究中通过 PCR 进行 HPV 诊断

• 批准号: 3423314
• 负责人: KEERTI V SHAH
• 金额: $ 8.25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-06-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3423314
• 关键词: Colombia; Europe; cancer risk; carcinoma; cervical /vaginal smear; cervix neoplasms; diagnosis procedure safety; human papillomavirus; human tissue; natural gene amplification; nucleic acid hybridization; polymerase chain reaction; virus DNA; virus classification; virus related neoplasm /cancer; western blottings

The polymerase chain reaction (PCR) technology which accomplishes in vitro enzymatic synthesis of DNA sequences has revolutionized molecular diagnosis. The aim of this investigation is to validate PCR diagnosis of human papillomaviruses (HPV) for epidemiologic studies, and to strengthen the virologic component of the International Agency for Research on Cancer (IARC)-sponsored case-control study of cervical cancer in Colombia and Spain. The IARC study is a comprehensive examination of all known and suspected risk factors of cervical cancer in two culturally related countries, one of which (Colombia) has a very high risk and the other (Spain) a very low risk of cervical cancer. It is proposed to examine a total of about 750 cervical scrapes obtained from cases of invasive cervical carcinomas and their population controls in Columbia and Spain. Stored aliquots of these specimens will be processed for PCR in Lyon in a laboratory which has no contact with HPVs. The PCR tests will be assembled in Baltimore using and HPV-free laboratory and reagents and equipment which have never been in contact with HPV. Amplification of HPV sequences will be achieved by the use of L1 consensus primers which are capable of synthesizing a segment of the L1 gene of many HPV types. The PCR products will be identified by Southern hybridization with type-specific end-labeled radioactive probes. The PCR procedure will be monitored for cross- contamination as well as for efficiency. Processing controls (containing SV40-transformed hamster cells) will be inserted among the specimens from the very beginning to monitor the possibility and extent of carry-over. Each PCR run will include negative controls (distilled water in place of specimen), positive controls (viral DNA) and amplification control (lambda DNA). n addition, subsets of the specimen will be tested for beta-globin gene amplification and for the frequency of sequences related to pBR322 plasmid. Five percent of the specimens which are positive in tests with L1 primers and five percent of negative specimens will be retested with a different set of primers (from the E6 region) to validate the initial diagnosis. The results of HPV diagnosis by PCR will be compared with the HPV diagnoses already made by dot blot and Southern hybridization assays to validate the technique for epidemiologic research. The PCR-based data will be added to other virologic data to examine the correlation of cytologic abnormalities in Pap smears with presence of HPV and to assess the contribution of different HPV types to the etiology of cervical cancer.

体外完成的聚合酶链式反应（PCR）技术 DNA序列的酶促合成彻底改变了分子生物学 诊断。 本研究的目的是验证 PCR 诊断 人乳头瘤病毒（HPV）的流行病学研究，并加强 国际癌症研究机构的病毒学部分 (IARC) 资助的哥伦比亚宫颈癌病例对照研究 西班牙。 IARC 研究是对所有已知和 两个文化相关的疑似宫颈癌危险因素 国家，其中一个（哥伦比亚）风险非常高，另一个 （西班牙）患宫颈癌的风险非常低。 建议审查一项 从侵入性病例中获得总共约 750 处宫颈擦伤 哥伦比亚和西班牙的宫颈癌及其人口控制。 这些样本的储存等分试样将在里昂的一个实验室中进行 PCR 处理 与 HPV 没有接触的实验室。 PCR测试将被组装 在巴尔的摩使用无 HPV 的实验室以及试剂和设备 从未接触过HPV。 HPV 序列的扩增将 通过使用 L1 共有引物来实现，该引物能够 合成多种 HPV 类型的 L1 基因片段。 PCR产物 将通过带有类型特异性末端标记的 Southern 杂交来鉴定 放射性探针。 PCR 程序将受到交叉监控 污染以及效率。 处理控制（包含 SV40 转化的仓鼠细胞）将被插入来自 从一开始就监测结转的可能性和程度。 每次 PCR 运行都将包括阴性对照（用蒸馏水代替 样本）、阳性对照（病毒 DNA）和扩增对照（λ 脱氧核糖核酸）。 此外，还将对样本的子集进行 β-珠蛋白测试 基因扩增以及与 pBR322 相关的序列频率 质粒。 百分之五的样本在 L1 测试中呈阳性 引物和百分之五的阴性样本将用 不同的引物组（来自 E6 区域）来验证初始 诊断。 PCR 诊断 HPV 的结果将与 HPV 诊断已通过斑点印迹和 Southern 杂交测定进行 验证流行病学研究的技术。 基于 PCR 的数据将 添加到其他病毒学数据中以检查细胞学数据的相关性 巴氏涂片检查中是否存在 HPV 异常，并评估 不同 HPV 类型对宫颈癌病因的贡献。