Development of method for integrated HPV16 detection in cervical carcinoma using multiplex quantitative PCR

Infection with high-risk HPV has been implicated as one of the major risk factors of cervical cancer. Integration of viral DNA into host DNA is essential for cervical carcinogenesis. This study was aimed to develop a multiplex real-time PCR to quantify Early gene 2 (E2 gene) and Early gene 6 (E6 gene) of HPV16 using Taqman probe. The ratio of E2 and E6 copy number was calculated to determine physical status of HPV16. The pure episomal form was expected to have an equivalent copy number of E2 and E6 gene giving rise to E2/E6 ratio of 1 whereas, viral integration resulted in less E2 than E6. The detection limit as well as precision of this developed method were obtained at 103 copies with CV of less than 10%. Cut off value of E2/E6 ratio for complete episomal form was found more than 0.83, whereas complete integration was expected to 0. This method was further analyzed with DNA from 15 pre-invasive or dysplasia lesions and 15 invasive cervical carcinoma tissues. The percentage of total integration form in invasive cases showed significantly higher than pre-invasive cervical lesions which obtained about 93%(14/15) and 66%(10/15), respectively (p value \< 0.05). The method described here is sensitive to assess the physical state as well as viral copy numbers, which suggests as a potential marker for disease progression. Furthermore, followed-up cases should be studied in large scale sample sizes in order to evaluate its potential as prognostic marker in cervical cancer.