School of Biological Sciences, University of Liverpool, Liverpool, UK.
Environ Microbiol. 2010 May;12(5):1194-204. doi: 10.1111/j.1462-2920.2010.02162.x. Epub 2010 Feb 9.
To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 10(31) tailed-bacteriophages are estimated to populate the globe. To address this discrepancy a culture-independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 x 10(4) to 6.5 x 10(6) ml(-1), with one in 10(3) free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation-dependent techniques, which grossly underestimate the abundance of viral entities.
为了确定在一个奶牛场中是否存在志贺毒素编码噬菌体（Stx 噬菌体）数量的季节性变化，我们对在 17 个月期间采集的水样进行了常规噬菌斑分析。噬菌体数量存在明显的季节性变化，在 6 月至 8 月之间达到峰值。将牛从牧场中移走会导致噬菌体数量减少，在冬季月份，没有检测到感染大肠杆菌的噬菌体，这一现象令人惊讶，因为据估计，全球有 10^31 个长尾噬菌体。为了解决这一差异，我们开发了一种基于定量 PCR 的非培养方法。设计了针对 Q 基因和 stx 基因的引物，这些引物可以准确且有区别地定量人工混合的 lambdoid 噬菌体群体。将这些引物组应用于噬菌斑分析未检测到噬菌体的水样，结果表明 lambdoid 噬菌体的数量范围为 4.7×10^4 至 6.5×10^6 ml^-1，其中每 10^3 个自由 lambdoid 噬菌体中就有一个携带志贺毒素操纵子（stx）。特定的分子生物学工具和有区别的基因靶标使我们能够对自然环境中的病毒群体进行计数，并且类似的策略可以替代现有的依赖于繁殖的技术，这些技术大大低估了病毒实体的丰度。
