Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB), CONICET, Instituto Universitario del Hospital Italiano (IUHI), Hospital Italiano (HIBA), Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Lett Appl Microbiol. 2021 Sep;73(3):308-317. doi: 10.1111/lam.13515. Epub 2021 Jun 11.
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.
对人类疱疹病毒 8 型（HHV-8）的 ORFK1 进行序列分析，可识别出 6 种主要亚型（A-F），它们与人类迁徙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 基因分型的最可靠方法。然而，它也存在挑战和局限性。在此，我们设计并验证了一种用于鉴定 HHV-8 ORFK1 亚型的单碱基延伸（SBE）方案。采用巢式聚合酶链反应（PCR）方案扩增包含 HHV-8 基因组中 360、406、465 和 527 位 4 个单核苷酸多态性的小 294-bp PCR 产物。最后，在 20 个先前通过系统发育分析进行基因分型的样本中开发并验证了多重 SBE 技术。该反应获得的图案可成功区分 ORFK1 亚型。在所有分析样本中，该方法获得的分型结果与“金标准”方法完全匹配。该方法可用于鉴定全球最常见的 HHV-8 亚型 A、B 和 C，以及其他亚型（D、E 和 F），具有可靠、快速、低成本的优势，特别适用于单管中低质量 DNA 样本的病毒基因分型，并且易于进行数据分析。
