HRM Analysis of HPV16 and HPV18 in Buccal Epithelial Cells

High resolution melt analysis (HRM) has recently been introduced to investigate the DNA integrity of PCR products. Because it takes less time than DNA sequencing and is reproducible. Oral cavity is a reservoir of human papillomaviruses (HPVs), which are associated with several oral cancers. Objective: The aim of this study was to employ the HRM to investigate HPV16 and HPV18 from the PCR products of buccal epithelial cells from 192 volunteers with normal oral mucosa. Methods: After DNA preparation, each sample was amplified with the HPV1003 and HPV1004 primers. All HPV positive samples were then processed to the real-time PCR using the specific primers from the E6 and E7 region of HPV16 and HPV18, respectively. The melting temperature and the shape of the melt cure of each amplicon were analyzed using the HRM. Results: A total of 47 samples were HPV-positive. Of these, 4 and 11 samples contained HPV16 and HPV18, respectively. HRM data revealed the shape of the melt curves was similar within the HPV16 and the HPV18 groups, implying the homozygosity of the DNA templates. However, each HPV group showed a temperature shift of 0.2-0.6°C in the melt peak amongst the positive samples. This suggests there should be a few nucleotide substitutions among the amplicons. Conclusions: The HRM is suitable for screening the similarity of PCR products, especially for a large sample size. However, one obvious disadvantage is that such tool is not able to identify the position of variations in the DNA sequences among the amplicons.