Jane Shen-Gunther1, Xin Yu. 1. Dept. of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, TX, USA. jane.shengunther@us.army.mil
Abstract
OBJECTIVE: To compare the analytical and clinical performance characteristics of 3 different primer sets targeting the human papillomavirus (HPV) L1 gene for detection and genotyping of HPV. METHODS: Liquid-based cytology was obtained prospectively from 90 colposcopy clinic patients. After automated extraction, cellular DNA was subjected to SYBR Green quantitative polymerase chain reaction (qPCR) using GP5+/6+ primers and conventional PCR with MY09/11 and FAP59/64 primers. The resulting SYBR Green counts and melting temperatures (T(m)) were used for quantification of HPV genomes and discrimination between presence and absence of amplicons. qPCR and PCR products were resolved by gel electrophoresis. HPV+amplicons were sequenced directly and BLAST® aligned for genotype identification. RESULTS: Of the 90 samples, 57 (63%) were qPCR+with a range of HPV viral load detected from 191 to 3.4 million genomes (~5 Log(10)). Only HPV-positives exhibited characteristic T(m) (75-80°C). The dynamic range of detection was similar for MY and FAP. Clinical net sensitivity was highest with simultaneous testing using all primer sets (93%) instead of individual primer pairs (GP (67%), MY (62%), FAP (49%)). Of the 27 HPV genotypes identified among 64 sequenced samples, the 3 most prevalent were HPV-16 (20%), -53 (9%), -31 (6%). The 4th rank included HPV-6, -33, -58, -66 (4.7% each). CONCLUSION: The performance characteristics of 3 leading PCR-based HPV assays revealed qPCR to be sensitive and specific for HPV detection and quantification. Parallel PCR testing using the 3 primers and direct sequencing offered the greatest clinical sensitivity and breadth of detection for known HPV types. Published by Elsevier Inc.
OBJECTIVE: To compare the analytical and clinical performance characteristics of 3 different primer sets targeting the human papillomavirus (HPV) L1 gene for detection and genotyping of HPV. METHODS: Liquid-based cytology was obtained prospectively from 90 colposcopy clinic patients. After automated extraction, cellular DNA was subjected to SYBR Green quantitative polymerase chain reaction (qPCR) using GP5+/6+ primers and conventional PCR with MY09/11 and FAP59/64 primers. The resulting SYBR Green counts and melting temperatures (T(m)) were used for quantification of HPV genomes and discrimination between presence and absence of amplicons. qPCR and PCR products were resolved by gel electrophoresis. HPV+amplicons were sequenced directly and BLAST® aligned for genotype identification. RESULTS: Of the 90 samples, 57 (63%) were qPCR+with a range of HPV viral load detected from 191 to 3.4 million genomes (~5 Log(10)). Only HPV-positives exhibited characteristic T(m) (75-80°C). The dynamic range of detection was similar for MY and FAP. Clinical net sensitivity was highest with simultaneous testing using all primer sets (93%) instead of individual primer pairs (GP (67%), MY (62%), FAP (49%)). Of the 27 HPV genotypes identified among 64 sequenced samples, the 3 most prevalent were HPV-16 (20%), -53 (9%), -31 (6%). The 4th rank included HPV-6, -33, -58, -66 (4.7% each). CONCLUSION: The performance characteristics of 3 leading PCR-based HPV assays revealed qPCR to be sensitive and specific for HPV detection and quantification. Parallel PCR testing using the 3 primers and direct sequencing offered the greatest clinical sensitivity and breadth of detection for known HPV types. Published by Elsevier Inc.
Authors: Jane Shen-Gunther; Chiou-Miin Wang; Graham M Poage; Chun-Lin Lin; Luis Perez; Nancy A Banks; Tim Hui-Ming Huang Journal: Clin Epigenetics Date: 2016-09-13 Impact factor: 6.551
Authors: Jane Shen-Gunther; Yufeng Wang; Zhao Lai; Graham M Poage; Luis Perez; Tim H M Huang Journal: BMC Genomics Date: 2017-03-14 Impact factor: 3.969