Human papillomavirus detection and typing using a nested-PCR-RFLP assay

Abstract

Background: It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Objectives: Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Methods: Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. Results: The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. Conclusion: The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing. Keywords: DNA probes, HPV, polymerase chain reaction, polymorphism, restriction, fragment length