Establishment of specifcations of the multiplex real-time PCR kit (NIHE-RealtimePCR hrHPV) for the detection of high-risk HPV genotypes

Abstract

High-risk human papillomavirus (HPV) genotypes are the leading causes of anogenital cancer, including cervical cancer. Screening these genotypes will potentially alleviate the disease burden that HPV causes. Therefore, NIHERealtime PCR hrHPV was developed based on the principle of multiplex real-time PCR. The objective of this study is to establish the specifcations of this assay in detecting and identifying 14 high-risk genotypes of HPV. The specifcations of this assay including cross-reactivity, sensitivity and specifcity, the limit of detection and stability, were evaluated with clinical samples, HPV positive control code 06/202 and 06/206 (National Institute for Biological Standards and Control) and in-house plasmids. Four different real-time systems and two commercial assays were applied for comparison. No cross detection of bacteria and viruses related to other genital-anal was observed. The sensitivity and the specifcity of this kit both reached 100% for HPV-18, 92.3% and 100% for HPV-16, and ranged from 90.0% - 100% for the other 12 high-risk genotypes (n = 92) with the detection limit of 5 copies/µl for HPV-16 and HPV-18, and 10 copies/µl for the other 12 high-risk genotypes HPV. This test was stable for up to 12 months under -20oC condition. Equivalent performance was demonstrated on all four real-time systems. Finally, the product was highly comparable with the Sacace kit, Italy (HPV Genotypes 14 Real-™ Quant, V67-100FRT), evaluated by the World Health Organization. The NIHE kit has the potential to be used in the early detection of HPV infection to improve control, prevention and treatment of HPV-related anogenital diseases.