Department of Genome Modifications and Carcinogenesis, Infection and Cancer Program, German Cancer Research Center, 69120 Heidelberg, Germany.
J Clin Microbiol. 2011 Oct;49(10):3560-7. doi: 10.1128/JCM.01173-11. Epub 2011 Aug 10.
Cutaneous human papillomaviruses (HPVs) are a heterogeneous, nonmonophyletic assembly, comprising about 50 characterized types and at least 133 isolates putatively representing new types. Their natural history of infection and potential association with nonmelanoma skin cancer are not well understood. Several PCR systems have been developed that amplify a broad spectrum of cutaneous HPVs. However, amplicon genotyping by sequencing or reverse line blot assays are complex and not well suited for high-throughput analyses. We developed a novel multiplex cutaneous papillomavirus genotyping (McPG) assay for 38 defined and 20 putative cutaneous HPVs of the beta, gamma, mu, and nu genera. Viral DNA was amplified by the use of a modified single-tube nested "hanging-droplet" FAP PCR. The amplifiable papillomavirus (PV) spectrum was enlarged by the use of 9 outer and 13 inner primers. Biotinylated PCR products were hybridized to type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads and analyzed using Luminex technology. Analytical sensitivity was analyzed for 38 defined HPVs and was ≤100 genome copies for all types. Integrated β-globin primers allow for simultaneous DNA quality control. McPG is characterized by high reproducibility (κ= 0.84, 95% confidence interval = 0.79 to 0.88), good concordance with the original nested FAP PCR, followed by sequencing (70.2% complete or partial agreement) when 322 skin biopsy DNA samples were analyzed, and improved ability to detect multiple infections (on average 2.5 HPV types per HPV-positive sample compared to 1.7 HPV types with nested FAP-PCR). In conclusion, McPG is a powerful tool for genotyping multiple cutaneous HPVs in a high-throughput format and is thus suitable for large-scale epidemiological studies.
皮肤人乳头瘤病毒（HPV）是一个异质的、非单系集合体，包括约 50 种特征明确的类型和至少 133 种假定的新类型分离株。它们的感染自然史及其与非黑色素瘤皮肤癌的潜在关联尚未得到很好的理解。已经开发了几种可扩增广谱皮肤 HPV 的 PCR 系统。然而，通过测序或反向线印迹分析对扩增子进行基因分型复杂，不适合高通量分析。我们开发了一种新的多重皮肤乳头瘤病毒基因分型（McPG）检测方法，用于 38 种明确和 20 种假定的β、γ、μ和 nu 属皮肤 HPV。使用改良的单管嵌套“悬滴”FAP-PCR 扩增病毒 DNA。使用 9 个外引物和 13 个内引物扩增可扩增的 HPV 谱。生物素化的 PCR 产物与特异性寡核苷酸探针杂交，连接到荧光标记的聚苯乙烯珠上，并使用 Luminex 技术进行分析。分析了 38 种明确 HPV 的分析灵敏度，所有类型的灵敏度均≤100 个基因组拷贝。整合的β-球蛋白引物允许同时进行 DNA 质量控制。McPG 的特点是具有高重复性（κ=0.84，95%置信区间=0.79 至 0.88），与原始嵌套 FAP-PCR 具有良好的一致性（70.2%的完全或部分一致），当分析 322 个皮肤活检 DNA 样本时，并且能够更好地检测多重感染（与嵌套 FAP-PCR 相比，平均每个 HPV 阳性样本检测到 2.5 种 HPV 类型，而 HPV 阳性样本为 1.7 种 HPV 类型）。总之，McPG 是一种高通量分析多种皮肤 HPV 的有力工具，因此适合大规模的流行病学研究。
