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A real-time PCR assay for HPV52 detection and viral load quantification
Juanita M Onyekwuluje1, Martin Steinau, David C Swan
1Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Division of High-Consequence Pathogens and Pathology, Chronic Viral Diseases Branch, Atlanta, GA 30333, USA. JOnyekwuluje@cdc.gov
Background:
Human papillomavirus (HPV) 52 is one of the most frequent high risk HPV types found in cervical and anal infections. Reliable and well characterized methods for HPV 52 detection are therefore of great importance. Unfortunately one of the most widely used commercially available HPV typing assays, the Roche Linear Array (LA), detects type 52 only through its XR probe which cross-reacts with types 33, 35 and 58 and fails to give unambiguous detection of HPV 52.
Methods:
To address this problem a real-time TaqMan PCR assay for HPV 52 targeting the E6/E7 region was developed and validated, which can be applied as robust duplex assay simultaneously detecting beta-globin as genomic control and reference or as highly sensitive single target detection assay.
Results:
Optimized primer and probe concentrations produced linear PCR amplifications over seven logs of targets (10(1) - 10(7)). The detection limit for HPV 52 was reproducibly at 10 copies per reaction for the duplex assay format and 5 copies for the single-plex format. The assay was very type-specific and no amplification signal was observed with 10(7) copies of the related HPV33, 35, and 58 DNA. Of 89 samples that tested unambiguously positive for HPV 52 in the LA, 75 were confirmed in the duplex format and 88 in the single-plex format. An additional 100 samples negative for HPV 52 in LA were all negative in both HPV 52 real-time PCR assay formats.
Conclusions:
These results indicate 92.6% and 99.5% accuracy relative to LA for the duplex and single-plex formats, respectively. In ongoing testing of 18,484 from various studies, 10.8% required the HPV52 TaqMan assay to unequivocally determine the status. Including the HPV 52 duplex assay provides the ability to monitor variations in the cell yield in various methods of sample collection and processing. This additional information is useful in QC monitoring of epidemiologic studies.
