Cervical cancer, which is caused by infection with oncogenic human papillomavirus (HPV) is the fourth most common cancer among women in worldwide. In particular, viruses 16 and 18 are known to account for about 70% of the causes of cervical cancer. Although the Pap smear is used as a primary method for cervical cancer screening, it is not possible to predict the potential risk of cancer due to the virus because it tests for cell deformation. Therefore, the need for HPV DNA testing for the early diagnosis of cervical cancer is increasing.
A new peptide nucleic acid (PNA)-assisted melting curve analysis technique is developed. Each genotype-specific PNA probe, which is conjugated with a fluorescent dye and a quencher, is used as a reporter in a real-time PCR reaction. A PNA probe can design relatively shorter binding sequence than DNA probe, so PNA probe can avoid sequence variation position on a gene. Furthermore, PNA probe showed bigger melting temperature difference than DNA probe when reporter probe bound to a single mismatch target. So, sequence variants on a target gene are easily distinguishable using melting curve analysis. Therefore, PNA-based reporter probe is very useful for multiplex detection in real-time PCR platform to identify a target gene with many sequence variants.
We have developed accurate and simple method to detect of HPV types within 3 hrs in one-tube. PNA probe-based fluorescence melting curve analysis technology in a real-time PCR system [PANA RealTyperTM HPV Screening Kit] is possible to detect 16 types of HPV [Genotyping types: 6, 11, 16, 18(Type associated with vaccine prescription) and Screening type: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68(HPV high risk)]. The PNA probes were designed to detect all variant genes for each HPV type. Using standard materials, each type of HPV identified the sensitivity of detection was 5 X 101 ~ 5X 103 copies. And there was no cross reaction with other HPV types.
PANA RealTyperTM HPV Screening kit is easily and rapidly can be detected HPV types, and it will be a useful and efficient method for detection and discrimination of HPV types in clinical diagnosis.