high-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes高分辨率快速融化的分子签名人乳头瘤病毒基因型的识别.pdfVIP

High-Resolution Melting Molecular Signatures for Rapid Identification of Human Papillomavirus Genotypes 1. 1,2. 1,3,4 1,2,5 Ta-Hsien Lee , Tzong-Shoon Wu , Ching-Ping Tseng * , Jiantai Timothy Qiu * 1 Graduate Institute of Biomedical Sciences, College of Medicine, Taoyuan, Taiwan, 2 Department of Biomedical Sciences, College of Medicine, Taoyuan, Taiwan, 3 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Taoyuan, Taiwan, 4 Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, 5 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan, Taiwan Abstract 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 a