Advances in metagenomics have revealed the ubiquity of single-stranded DNA (ssDNA) phage belonging to the subfamily Gokushovirinae in the oceans; however, the abundance and ecological roles of this group are unknown. Here, we quantify gokushoviruses through adaptation of the polony method, in which viral template DNA is immobilized in a gel, amplified by PCR, and subsequently detected by hybridization. Primers and probes for this assay were designed based on PCR amplicon diversity of gokushovirus major capsid protein gene sequences from a depth profile in the Gulf of Aqaba, Red Sea sampled in September 2015. At ≥95% identity, these 87 gokushovirus sequences formed 14 discrete clusters with the largest clades showing distinct depth distributions. The application of the polony method enabled the first quantification of gokushoviruses in any environment. The gokushoviruses were most abundant in the upper 40 m of the stratified water column, with a subsurface peak in abundance of 1.26 × 10⁵ viruses ml⁻¹. These findings suggest that discrete gokushovirus genotypes infect bacterial hosts that differentially partition in the water column. Since the designed primers and probe are conserved across marine ecosystems, this polony method can be applied broadly for the quantification of gokushoviruses throughout the global oceans.

中文翻译：

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采用 polony 技术量化 Gokushovirinae，一组多样化的单链 DNA 噬菌体

宏基因组学的进展揭示了属于Gokushovirinae亚科的单链 DNA (ssDNA) 噬菌体的普遍存在在海洋中；然而，这一群体的丰度和生态作用尚不清楚。在这里，我们通过采用 polony 方法来量化 gokushoviruses，其中病毒模板 DNA 固定在凝胶中，通过 PCR 扩增，然后通过杂交检测。该测定的引物和探针是根据 2015 年 9 月红海亚喀巴湾深度剖面中 gokushovirus 主要衣壳蛋白基因序列的 PCR 扩增子多样性设计的。在 ≥95% 的同一性下，这 87 个 gokushovirus 序列形成 14 个离散具有最大进化枝的集群显示出明显的深度分布。polony 方法的应用能够在任何环境中首次量化 gokushoviruses。gokushoviruses 在分层水柱的上部 40 m 中最丰富，⁵种病毒 ml ^-1。这些发现表明，离散的 gokushovirus 基因型感染在水柱中差异分配的细菌宿主。由于设计的引物和探针在海洋生态系统中是保守的，因此这种群体方法可以广泛应用于全球海洋中的 gokushoviruses 的量化。