Opment and Optimization of PCR Assay for )n of High Risk Human Papillomavirus Types

Abstract

High risk human papillomaviruses (HPV) are the major causes for development of cervical cancer in females and anal cancer in both male and female populations all over the world. Considerable increase in morbidity and mortality rates due to HPV infections over the past decade, the inconvenience of applying many identification methods to confIrm presence of different high risk HPV types (HPV HRT), and lack of medications for successful treatment for HPV infections prompted us to develop a PCR assay that can detect a number of high risk HPV types in a single reaction. We describe here i) the development of an efficient, fast, non-toxic and cheap method for HPV DNA extraction, ii) universal primer development for high risk HPV types and iii) optimization of polymerase chain reaction (PCR) assay for high risk HPV types detection. For this purpose, 50 cervical secretion samples were collected from females reporting at three tertiary care hospitals in Rawalpindi and Islamabad. HPV DNA was extracted from these samples using a self-developed novel CT AB-Guanidine Isothiocyanate method. Two sets of primers, HPV HRT-l for detection ofHPV types 16, 31 , 33, 35, 52 and 58, while HPV HRT-2 for detection of HPV types 18, 45, 56, 59, 68 and 69 were designed using MAFFT and MEGA5 bioinformatics tools and commercially synthesized. Optimization of PCR conditions resulted in identification of two HPV positive samples on gel electrophoresis, one positive for HPV HRT-l and the other positive for HPV HRT-2. Sequencing of one of the amplified PCR product confmned the identification of relative HPV type. Our results show the development of a promising, efficient and economical method for HPV DNA extraction and identifIcation which can confirm the presence of high risk HPV types in cervical secretions. Our work is of specific importance from research point of view, where HPV DNA obtained from cervical secretions is often less in quantity and difficult to work with. Apart from its application prior to type specific identification of high risk HPV types, this newly developed PCR assay will also ease the process of HPV testing in Pakistan, where lack of health facilities restrict HPV testing in general hospitals.