RESUMEN
Objective To establish a method to detect viral integrity of human papillomavirus in women cervical HPV infection. Methods We amplified E6/E7 gene and E2 gene of HPV16,then inserted them into a plasmid containing single copy HBB gene. HPV16 infected cervical epithelium samples were screened out by genotyping with RDB of flow-through hybridization assay.Fluo-rescence quantitive PCR data of HBB,viral E2 gene and viral E6 gene of all samples were standardized by compared with respective parameters of the plasmid.The ratio IHPV and CHPV were calculated to find out E2 gene disruption and viral copies per cell in the cer-vical samples,respectively.Results The plasmid constructed for standardization was proved effective to make the FQ-PCR data of E2 gene,E6 gene and HBB gene comparable.Thirty-seven HPV16 positive cervical epithelium samples included 22 cases from women whose TCT were normal,and 15 cases from women who confirmed HIL/CIN 2-3 or above through colposcopic examina-tion plus biopsy.Fifteen samples were detected E2 gene disruption,including 10 HIL/CIN 2-3 or above samples and 5 TCT normal samples.E2 gene integrity in different groups were statistically significant different(P <0.05).The average viral copies per cell dis-played a significant decline along with E2 gene disruption(P <0.05).Conclusion The tandem single copy gene plasmid standard-ized methord for the detection of E2 gene disruption caused by viral integration in HPV16 infected cervical cells is feasible and effec-tive.
RESUMEN
Objective To investigate the distribution of human papillomavirus (HPV) type 16 in women cervical infection in eastern Guangzhou, polymorphism of E6/E7 gene and association of gene dosage with disease progression. Methods Flow-through hybridization and gene chips were applied in HPV sub-type identification to screen out HPV-16 positive samples from cervical epithelium samples. HPV-16 E6/E7 gene was amplified through PCR with specific primers. The PCR products were cloned into pMD18-T vectors and fragments were determined through sequencing. Polymorphism analysis were performed through align-ment tools. Fluorescence quantitive PCR were used for the detection of viral E6 gene and L1 gene. Results Thirty-six (4.5%) HPV-16 positive samples were screened out through flow-through hybridization from 806 cervical epithelium samples. HSIL and above happened in 18 (50.0%) of the 36 HPV-16 positive patients. Within E6/E7 gene sequences from 7 selected samples, we found 15 sites with variances and 8 of them would cause coding amino acid change. HIL group (A, 11 cases) and LSIL group (B, 14 cases) possess significantly different gene dosage of both viral E6 gene and LI gene (P <0.05). The ratios of L1/E6 be-tween the 2 groups was not significantly different(P=0.19). Conclusion HPV-16 cervical infection oc-curs in 4.5% women (17-62 years old) in eastern Guangzhou. HIL or above accompany with half of the HPV 16 infected women. Viral load is probably associated with cervical HSIL, though L1/E6 ratios do not suggest viral integration.