Cancer Gene Therapy Group, Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, k.1, LV-1067 Riga, Latvia.
Viruses. 2023 Apr 26;15(5):1060. doi: 10.3390/v15051060.
The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications.
病毒定量对于研究和临床应用都是必要的。现有的 RNA 病毒定量方法存在一些缺点，包括对抑制剂的敏感性和标准曲线生成的必要性。本研究的主要目的是开发和验证一种使用液滴数字 PCR（ddPCR）定量重组、复制缺陷型 Semliki Forest 病毒（SFV）载体的方法。该技术使用针对插入的转基因、SFV 基因组的 nsP1 和 nsP4 基因的各种引物，证明了其稳定性和可重复性。此外，通过优化退火/延伸温度和病毒：病毒比例，成功测量了两种类型的复制缺陷型重组病毒颗粒混合物中的基因组滴度。为了测量感染性单位，我们开发了一种单细胞 ddPCR，将整个感染细胞添加到液滴 PCR 混合物中。研究了细胞在液滴中的分布，并使用 β-肌动蛋白引物进行定量标准化。结果，定量了感染细胞的数量和病毒感染性单位。潜在地，所提出的单细胞 ddPCR 方法可用于临床应用中定量感染细胞。
