Division of the Aquatic Biology and Ecology, Center for Marine Environmental Studies (CMES), Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790–8577, Japan.
Microbes Environ. 2010;25(3):211-5. doi: 10.1264/jsme2.me10117.
The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method to be a potential tool for environmental monitoring of cyanophage infections. In this field survey, the numbers of infected M. aeruginosa cell populations estimated from cyanophage numbers were low at 0.01-2.9 cells mL(-1). The highest relative abundance of phage g91 RNA (10(-2) per rnpB transcript) was at about the same levels of expression as laboratory-based growth data for Ma-LMM01 (estimated density of infected host cells: 10(5) cells mL(-1)); and was observed when cyanophage numbers rapidly increased (as well as a decrease in host cell numbers). Quantification of cyanophage numbers is important to understand ecological relationships between the phage and its hosts. Our data suggest the quantification of phage gene transcripts within a natural host cell population to be a strong tool for investigating the quantitative effects of phage lysis during infection of the host population.
本研究旨在开发一种定量实时逆转录聚合酶链反应（real-time RT-PCR）检测方法，以检测和定量淡水池塘中感染铜绿微囊藻细胞内的噬藻体 mRNA。基于实验室的研究数据表明，在培养过程中噬藻体数量增加之前，宿主细胞内噬藻体 g91 mRNA 的相对丰度增加。这种转录模式表明了病毒感染的动力学，提示实时 RT-PCR 方法可能成为环境中监测噬藻体感染的一种潜在工具。在本野外调查中，根据噬藻体数量估计的感染铜绿微囊藻细胞群体数量很少，在 0.01-2.9 个细胞/mL 之间。噬藻体 g91 RNA 的最高相对丰度（每 rnpB 转录物 10(-2)）与 Ma-LMM01 的基于实验室的生长数据的表达水平相当（估计的感染宿主细胞密度：10(5) 个细胞/mL）；并且在噬藻体数量快速增加时（以及宿主细胞数量减少时）观察到。定量噬藻体数量对于了解噬菌体与其宿主之间的生态关系非常重要。我们的数据表明，定量分析天然宿主细胞群体内噬菌体基因转录本是研究宿主群体感染过程中噬菌体裂解对定量影响的有力工具。
