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.
| Guerrero, E; Daniel, R W; Bosch, F X et al. (1992) Comparison of ViraPap, Southern hybridization, and polymerase chain reaction methods for human papillomavirus identification in an epidemiological investigation of cervical cancer. J Clin Microbiol 30:2951-9 |
