Abstract

Introduction: Higher viral load in women with chronic Human Papillomavirus type 16 (HPV 16) infection and integration of the viral genome into the cell's chromosomes associates with an increased prevalence of premalignant/malignant lesions in the cervix. The present study describes the development of simultaneous measurement of viral load and physical status for the HPV16 genome by the Multiplex Real-Time PCR method. Methods: In this study, conserved genes (E6 and E2) of HPV16 were extracted from the CaSki cell line and, after PCR amplification, cloned in bacterial plasmid pJET1.2/blunt cloning vector. In addition, recombinant cellular RnaseP gene plasmid was used as an internal amplification control gene. Results: Based on our findings, the E2 and E6 genes of the HPV16 virus and the cellular RnaseP gene are identified with high efficiency. Besides, the results of the Real-time PCR showed a high R2 coefficient that reflects the linearity of the standard curve. The results of the E2/E6 ratio of CaSki cells (E2/E6 ratio=0.15) indicated the presence of both integrated and episomal forms in this cell line. Conclusion: According to the pre-determined acceptance criteria, the diagnostic panel designed to measure the viral load and the physical status of the HPV16 genome using the Multiplex Real-Time PCR method has appropriate sensitivity and specificity. © 2023, Shahroud University of Medical Sciences. All rights reserved.