High-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes.
BACKGROUND
Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.


METHODS
Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.


RESULTS
A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215-221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.


CONCLUSIONS
This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.
Graduate Institute of Biomedical Sciences, College of Medicine, Taoyuan, Taiwan.
Ta-Hsien
TH
Lee
Tzong-Shoon
TS
Wu
Ching-Ping
CP
Tseng
Jiantai Timothy
JT
Qiu
D052685:Alphapapillomavirus / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D005814:Genes, Viral; D005838:Genotype; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid