Multiplex PCR assay for the rapid identification of human papillomavirus genotypes 16, 18, 45, 35, 66, 33, 51, 58, and 31 in clinical samples

The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has lead to the development of a variety of molecular assays for HPV detection. The present study focused on the development of a simple, sensitive and cost-effective HPV genotyping method based on multiplex PCR methodology that could be easily performed in small laboratories. Three multiplex PCR assays were developed to identify the HPV genotypes 16, 18, 45, 35, 66, 33, 51, 58, and 31 together with an internal control. The method was established by designing nine type-specific primer sets that target conserved regions of the L1 gene. The assay was applied using HPV-positive cervical specimens, and cloning and sequencing of all of the amplicons that were generated were performed to examine the specificity of the newly designed primers. Moreover, an experimental cutoff value was determined through reconstitution experiments using HPV DNA plasmids. Amplicons of expected size were obtained, while cloning and sequencing of PCR products confirmed the genomic specificity of the amplicons. The sensitivity of this method was determined to be 10 copies of each individual HPV genotype per test. Multiple and single HPV infections were documented in 42.2 % and 57.8 % of cervical specimens, respectively. The most prevalent HPV genotype was HPV16, followed by HPV18, HPV66 and HPV51. The present multiplex PCR assay is a simple method with high specificity and sensitivity that can be applied in clinical or epidemiological analyses for rapid identification of the most clinically important HPV genotypes present in cervical intraepithelial neoplasias.