Sequence analysis of the ORFK1 of human herpesvirus type 8 (HHV-8) allows the identification of six major subtypes (A–F), which are related to human migrations and the clinical progression of Kaposi's sarcoma. Sequencing and subsequent phylogenetic analysis of ORFK1 is considered to be the most reliable method for HHV-8 genotyping. However, it exhibits challenges and limitations. Herein, we designed and validated a single base extension (SBE) protocol for characterization of HHV-8 ORFK1 subtypes. A nested polymerase chain reaction (PCR) protocol was carried out to amplify a small 294-bp PCR product encompassing four single nucleotide polymorphisms at positions 360, 406, 465 and 527 of the HHV-8 genome. Finally, a multiplex SBE technique was developed and validated in 20 samples previously genotyped by phylogenetic analysis. The patterns obtained in this reaction could successfully discriminate between ORFK1 subtypes. The typing results obtained completely matched with those of the ‘gold standard’ method in all analysed samples. This method can reliably identify HHV-8 subtypes A, B and C, which are the most prevalent ones worldwide, and the remaining subtypes (D, E and F). SBE can be useful as an efficient, rapid and low-cost screening method for viral genotyping in a single tube, particularly samples with low-quality DNA, and with easy data interpretation.

中文翻译：

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一种使用单碱基延伸进行人类疱疹病毒 8 型亚型表征的简单快速方法

人类疱疹病毒 8 型 (HHV-8)的ORFK1的序列分析允许鉴定六种主要亚型 (A-F)，它们与人类迁移和卡波西肉瘤的临床进展有关。ORFK1 的测序和随后的系统发育分析被认为是 HHV-8 基因分型最可靠的方法。然而，它表现出挑战和局限性。在此，我们设计并验证了用于表征 HHV-8 ORFK1的单碱基扩展 (SBE) 协议亚型。进行巢式聚合酶链反应 (PCR) 协议以扩增包含 HHV-8 基因组第 360、406、465 和 527 位的四个单核苷酸多态性的 294 bp PCR 小产物。最后，在先前通过系统发育分析进行基因分型的 20 个样本中开发并验证了多重 SBE 技术。在该反应中获得的模式可以成功区分ORFK1亚型。在所有分析样品中获得的分型结果与“金标准”方法的结果完全匹配。这种方法可以可靠地识别 HHV-8 亚型 A、B 和 C，它们是世界上最普遍的亚型，以及其余的亚型（D、E 和 F）。SBE 可用作在单管中进行病毒基因分型的高效、快速和低成本筛选方法，尤其是具有低质量 DNA 且易于数据解释的样本。