Department of Microbiology, Savitribai Phule Pune University, Ganeshkhind, Pune, 411007, India; Food Technology Division, Bhabha Atomic Research Centre, Mumbai, 400085, India.
Molecular Biology Division, Bhabha Atomic Research Center, Mumbai, 400085, India.
Virus Res. 2019 Apr 2;263:184-188. doi: 10.1016/j.virusres.2019.02.005. Epub 2019 Feb 12.
The phage taxonomy is primarily based on the morphology derived from Transmission Electron Microscopic (TEM) studies. TEM based characterization is authentic and accepted by scientific community. However, TEM based identification is expensive and time consuming. After the phage isolation, before analysis TEM, a DNA based rapid method could be introduced. The DNA based method could dramatically reduce the number of samples analyzed by TEM and thereby increase the speed and reduce the cost of identification. In the present work, four environmental phage isolates were identified based on TEM studies and genome size. The identification of these four phages was validated using DNA based method. The taxon-specific DNA markers were identified through multiple sequence alignments. The primers were designed at conserved genes (DNA polymerase or integrase) of 4 different phage taxa viz. family Ackermannviridae, genus Jerseyvirus, genus T4virus, and genus P22virus. These primers were evaluated using both in vitro and in silico approach for the amplification of the target taxons. Majority of the primer sets were found to amplify member species of the targeted taxa in vitro. In In silico analysis, six primer sets intended for identification of family Ackermannviridae showed positive amplification of ≥86.7% classified species. Further, the primers targeting the genus Jerseyvirus and T4virus showed the amplification of 53.8% and ≥84.6% species, respectively. The present work is a case study performed to explore the possibility of use of taxon-specific primers for identification and taxonomic studies of newly isolated phages to supplement the TEM.
噬菌体分类主要基于透射电子显微镜（TEM）研究得出的形态学特征。基于 TEM 的特征分析是真实的，并且被科学界所接受。然而，TEM 鉴定既昂贵又耗时。在噬菌体分离后，在进行 TEM 分析之前，可以引入基于 DNA 的快速方法。这种基于 DNA 的方法可以大大减少需要通过 TEM 分析的样本数量，从而提高鉴定速度并降低成本。在本工作中，根据 TEM 研究和基因组大小对四个环境噬菌体分离株进行了鉴定。使用基于 DNA 的方法验证了这四种噬菌体的鉴定。通过多序列比对鉴定了分类特异性 DNA 标记。设计了针对四个不同噬菌体类群（Ackermannviridae 科、Jerseyvirus 属、T4virus 属和 P22virus 属）的保守基因（DNA 聚合酶或整合酶）的引物。通过体外和计算机模拟两种方法评估了这些引物对目标分类群的扩增效果。大多数引物在体外对目标分类群的成员物种都具有扩增作用。在计算机模拟分析中，用于鉴定 Ackermannviridae 科的六个引物集对 86.7%以上的分类物种显示出阳性扩增。此外，针对 Jerseyvirus 属和 T4virus 属的引物分别显示出对 53.8%和≥84.6%的物种的扩增。本工作是一项案例研究，旨在探索使用分类特异性引物对新分离噬菌体进行鉴定和分类学研究的可能性，以补充 TEM 的不足。
