A sensitive and quantitative method for the determination of number of HPV 16 DNA copies by using the competitive...

The infectivity of human papillomavirus (HPV) is closely linked to the characteristics of the epithelial lesions that it induces in the uterine cervix. In a previous study of ours, we reported that the incidence of positivity for HPV DNA increases as lesions progress toward cervical carcinoma. However, the incidence of positivity did not increase during the process of tumorigenesis from severe dysplasia to invasive carcinoma. These findings suggest that patients with invasive carcinoma had much fewer DNA copies of HPV in host cells. Based on these findings, we have prepared a HPV16 DNA standard (DNA standard) with an EcoRI restriction site by using a polymerase chain reaction (PCR) technique and site-directed mutagenesis. We have also developed a sensitive quantitative method based on PCR that uses primers and competition between the patient's HPV16 DNA and an internal DNA standard for the determination of numbers of copies of HPV16 DNA. In this study, uterine cervical tissues obtained from 12 patients with invasive carcinoma were studied using competitive PCR (CPCR) to determine the number of HPV16 DNA copies. We found that the number of copies in the tissues of patients with invasive varied between 5 x 10(6) to 4 x 10(9)/micrograms DNA.