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Oncogenic human papillomavirus (HPV) is the most important risk factor for cancer of the uterine cervix and a subgroup of head and neck cancers. Viral load has been associated with persistence of infection, whereas integration of HPV into the host cell genome is associated with transition to invasive disease. Viral integration is frequently correlated with loss of viral E2 and gain of the telomerase-related genesTERCandTERT. The objective of this study was to develop a rapid and sensitive multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous analysis of viral load, integration and copy number gain ofTERCandTERTin HPV16/18-associated lesions. The performance of the assay was tested for HPVvs. human gene copy number ratios ranging from 0.1 to 100 and for percentages of integration ranging from 0 to 100%. The model systems used include plasmid mixtures and the HPV-positive cell lines SiHa, HeLa and CaSki described to contain a range of 2–600 viral copies per cell. In samples with low-viral load, viral integration can be reliably determined when more than 30% of the virus is integrated. Gain of the telomerase-related genes in the cell lines as determined by our MLPA assay was in accordance with data reported in the literature. Our study demonstrates that within a single MLPA-reaction viral type, load, integration and gain ofTERCandTERTcan be reliably determined, which will improve risk assessment for patients suspected for HPV infection.