This study investigates the interaction between Saccharibacteria and Actinomyces through PCR amplification techniques. The methodology focuses on isolating and visualizing the genomic DNA of Saccharibacteria within a binary culture.
Begin with a Saccharibacteria-infected Actinomyces culture.
Saccharibacteria, an ultrasmall parasitic bacterium, and its host form a binary culture.
Add an aliquot of the infected culture to a PCR tube containing amplification reagents with electrophoresis tracking dye and Saccharibacteria-specific primers.
Place the tube in a thermocycler with set PCR conditions.
During initial denaturation, heat lyses Saccharibacteria, releasing genomic DNA. Actinomyces remain intact due to rigid cell walls, limiting host DNA release.
The heat then denatures the Saccharibacteria DNA strands and activates Taq polymerase.
During annealing, the primers bind to complementary sequences in the Saccharibacteria genome.
In extension, Taq polymerase adds dNTPs to the primers, synthesizing new DNA strands.
Load the amplified PCR product onto an agarose gel pre-stained with a dye that binds to the DNA fragments.
Run electrophoresis and visualize the gel under UV light.
A Saccharibacteria-specific amplicon band confirms host–parasite interaction in the binary culture.
Aliquot 24 microliters of the PCR master mix into a 0.2 milliliter PCR tube. At one microliter of saccharibacteria infected culture, and place the tube in a Thermo cycler.
Set the PCR program as described in the text manuscript. Load and run the PCR products on a 1% Agros gel. A band of approximately 600 bases will confirm the presence of saccharibacteria.
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