Improved detection of human papillomavirus harbored in healthy skin with FAP6085/64 primers

https://doi.org/10.1016/j.jviromet.2013.06.026Get rights and content

Highlights

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    The FAP6085/64 primer set was first evaluated in this study.

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    The FAP6085/64 combination was much more sensitive than the original primer sets using HPV plasmids and 90 skin samples as templates.

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    The FAP6085/64 showed good performance in detecting 2807 skin samples from healthy population.

Abstract

FAP59/64, FAP6085/6319, and CUT primer sets were designed for detecting cutaneous HPV and have been used in many clinical and epidemiology studies. The FAP6085/64 primer set was first evaluated in this study and the FAP6085/64 combination was found to be much more sensitive than all three original primer sets by using HPV plasmids as a template. To confirm further the effectiveness of the FAP6085/64 primer set in human DNA templates, 90 palmar exfoliated cell DNA samples were used to detect the cutaneous HPV by both the FAP59/64 and FAP6085/64 primer sets. The overall proportion of HPV detection in those skin samples was 77.8% (70/90) using FAP6085/64, as compared to 55.6% (50/90) using FAP59/64. The FAP6085/64 primer set was also applied in a population based study. The proportion of HPV detection was 73.96% (2076/2807) in skin samples collected from healthy individuals, and a total of 336 different PV types were found. Sixty (17.9%) of them were fully characterized HPV types, 127 (37.8%) were putative HPV types which had been described previously, 149 (44.3%) were novel putative HPV types, and two animal PVs were also detected. These results suggest that the FAP6085/64 primer set was sensitive and effective for detection of cutaneous HPV in healthy skin samples.

Introduction

Papillomaviruses (PVs) are a large and highly diverse group of small, non-enveloped, icosahedral-shaped viruses containing a double-stranded DNA genome of approximately 8 kb (de Villiers et al., 2004). PVs infect the basal layer of cutaneous or mucosal epithelial cells of a dozen vertebrate species with strict host and trophic specificity. Human papillomavirus is one of the most important groups of the PV family and it can be divided roughly into two groups based on tropism, cutaneous HPV types and mucosal HPV types that infect the skin and mucosal tissues respectively (de Villiers, 1989). High-risk mucosal HPV types cause cervical cancer and are associated with numerous other cancers such as head and neck cancer (zur Hausen, 2009). However, the role of cutaneous HPV types in pathogenesis remains unclear. HPVs found in squamous cell carcinoma (SCC) of the skin are mostly HPV 5 or 8 and sometimes HPV 14, 17, 20 or 47 and these are regarded as potential high-risk types (Pfister, 2003), while most other cutaneous HPVs cause only benign lesions such as epidermodysplasia verruciformis (EV) (Akgul et al., 2006).

Human skin harbors a very large spectrum of HPV types, and most of these were unknown previously (Antonsson et al., 2003). Most of the recently reported novel HPV types have been isolated from skin samples (de Villiers and Gunst, 2009, Kohler et al., 2011, Kovanda et al., 2011) and belong to the Beta or Gamma papillomavirus genus. Ubiquity and multiplicity are two noteworthy characteristics of cutaneous PV infection (Antonsson and Hansson, 2002) as compared with mucosal PV infection. Although the genomic organization of mucosal and cutaneous types is similar, the association between cutaneous HPV infection and human malignancy remains open to question (Pfister, 2003). Epidemiology and evolutionary biologic studies are needed to explore the reasons underlying this significant difference.

The development of PCR methods using general primers for amplification of a broad range of HPV types has had a major impact on the molecular epidemiology of HPV. Many different primer sets targeting the L1 ORF have been designed and used for the detection of a broad range of HPV types in healthy skin as well as in non-melanoma skin cancers (Gheit et al., 2007, Harwood et al., 1999, Sasagawa and Mitsuishi, 2012). The FAP primer set (Forslund et al., 1999, Forslund et al., 2003) is the most used one. There are two pairs of FAP primers, known as FAP59/64, and FAP6085/6319. FAP59/64 was originally used as outer primers and FAP6085/6319 as inner primers in a single tube nested PCR (Forslund et al., 2003). The CUT primer set (Chouhy et al., 2010) was described recently and it was used in single tub with an identical primer nested PCR. All of the three sets above have been proven to have good ability to amplify the cutaneous HPV L1 gene. However, the laboratory work necessary for nested PCR is a challenge in projects of large sample size, and a single round of PCR is preferable for this purpose.

In the present study, all of the available cutaneous HPV sequences were collected and aligned with FAP59, FAP64, FAP6085, 6319 and CUT primers, and FAP6085 and FAP64 showed greater similarity to the nucleotide sequence of the reference HPV. FAP6085 and FAP64 were then chosen as a new primer set and the amplification efficiency of this set was compared with three original primer pairs in single round PCR. This new primer combination was then used to test 2807 samples collected from healthy individuals.

Section snippets

FAP outer primer (FAP59/64) system (A)

PCR amplification using the FAP59/64 primer was performed as described previously (Forslund et al., 1999) with minor modifications. 25 μl of PCR mixture containing 1 μl of HPV plasmid DNA or 5 μl of DNA extracted from exfoliated cell samples of skin, 0.75 μM each of FAP59 (5′-TAACWGTIGGICAYCCWTATT-3′) and FAP64 (5′-CCWATATCWVHCATITCICCATC-3′) primer, 200 μM of each dNTPs (Fermentas, Vilnius, Lithuania), 3.5 mM MgCl2, 0.625 units of hot-start Taq polymerase (Qiagen, Hilden, Germany), and hot-start

Generality and sensitivity of the four PCR primer sets

HPV 5, 8, 37 and 49 were selected as the first panel for testing the A–D primer sets. The size of the HPV amplicons were 478 bp for primer A (FAP59/64), 235 bp for primer B (FAP6985/6319), 370 bp for primer C (CUT), and 377 bp for primer D (FAP6085/64). Using 104 or more copies of HPV 5, 8, 37, and 49 plasmids as the templates, the expected fragments were amplified successfully by the corresponding primer sets. However, when the template copy number decreased to 5000/reaction, the amplification

Discussion

The present study demonstrated that the FAP6085 and FAP64 primer combination (FAP6085/64) was more sensitive than other primer sets in ‘single round’ PCR, and this combination was also highly effective for identification and characterization of HPV infection in normal skin samples.

The conventional two-step nested PCR method or single tube nested ‘hanging droplet’ PCR method which was developed recently may have better sensitively than single round PCR. However, any nested PCR method is prone to

Acknowledgments

This work was supported by the grants from the 973 project (201202014), the National Natural Sciences Foundation of China (30930102), the Beijing Natural Science Foundation (7100001), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20110001110037).

We would like to thank Dr. Michael A. McNutt for editing and correction of this manuscript.

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