Related Experiment Video
Updated: Apr 18, 2026
Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
Published on: November 5, 2020
Development of a real-time PCR assay for the quantification of Ma-LMM01-type Microcystis cyanophages in a natural
S Kimura-Sakai1, Y Sako, T Yoshida
1Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, Japan.
Unlabelled:
Microcystis aeruginosa forms toxic cyanobacterial blooms throughout the world where its infectious phages are thought to influence host population dynamics. To assess the cyanophage impact on the host dynamics, we previously monitored Ma-LMM01-type phage abundance using a real-time PCR with a primer set designed based on the sequence of Microcystis phage Ma-LMM01; and we estimated the phage-infected host cell abundance. However, a recent study shows the Ma-LMM01 g91 gene sequence belongs to the smallest group, group III, of the three genotype groups, suggesting Ma-LMM01-type phage abundance was underestimated. Therefore, to re-evaluate the effect of Ma-LMM01-type phages on their hosts, we monitored the abundance of Ma-LMM01-type phages using real-time PCR with a new primer set designed based on the sequences of genotype groups I-III. We found phage abundance between 10(3) and 10(4) ml(-1) using the new primer set in samples where previously these phages were not detected using the old primer set. The frequency of Ma-LMM01-type phage-infected cells to Ma-LMM01-type phage-susceptible host cells may be as high as 30%, suggesting the phages may occasionally affect not only shifts in the genetic composition but also the dynamics of Ma-LMM01-type phage-susceptible host populations.
Significance And Impact Of The Study:
Phages are one of the factors that may control the ecology of their host blooms. Therefore, it is essential to estimate phage abundance to understand phage impact on host populations. A real-time PCR assay was improved to detect a larger range of Microcystis cyanophages in natural surroundings where no phages were detected using a previous method by re-designing a new primer set based on sequences from three Ma-LMM01-type phage genetic groups. The new method allows us to determine the distribution, dynamics and infection cycle of the phage to help understand the interaction between the phages and the hosts.
Insights
Improved real-time PCR methods reveal previously undetected cyanophage (Ma-LMM01-type) abundance, suggesting phages significantly impact Microcystis bloom dynamics and host populations. This finding is crucial for understanding cyanobacterial bloom control.
Area of Science:
- Environmental Microbiology
- Virology
- Ecology
Background:
- Toxic cyanobacterial blooms, particularly Microcystis aeruginosa, are globally prevalent.
- Infectious phages are hypothesized to influence cyanobacterial population dynamics.
- Previous phage monitoring underestimated Ma-LMM01-type phage abundance due to limited primer set coverage.
Purpose of the Study:
- To re-evaluate the ecological impact of Ma-LMM01-type phages on Microcystis populations.
- To develop a more comprehensive method for quantifying Ma-LMM01-type phage abundance.
- To assess the frequency of phage-infected host cells in natural environments.
Main Methods:
- Real-time PCR was employed to quantify phage abundance.
- A new primer set was designed based on Ma-LMM01-type phage genotype groups I-III for improved detection.
- Phage-infected host cell abundance was estimated in relation to susceptible host populations.
Main Results:
- The improved real-time PCR assay detected Ma-LMM01-type phages (10^3-10^4 ml^-1) in samples previously yielding no detection.
- The frequency of infected cells relative to susceptible hosts reached up to 30%.
- These findings suggest phages can significantly influence host population dynamics and genetic composition.
Conclusions:
- Phages are critical factors controlling cyanobacterial bloom ecology.
- Accurate phage abundance estimation is essential for understanding their impact on host populations.
- The improved real-time PCR assay enhances the detection of Microcystis cyanophages, aiding in the study of phage-host interactions and infection cycles.
