This article details a method for enumerating bacteriophages using quantitative polymerase chain reaction (qPCR). The process involves preparing a qPCR reaction cocktail, adding heat-treated bacteriophages, and analyzing the resulting fluorescence to quantify bacteriophage particles.
To enumerate bacteriophages via quantitative polymerase chain reaction, qPCR, obtain a qPCR reaction cocktail containing Taq DNA polymerase, primers, deoxynucleotide triphosphates — dNTPs — and fluorescent reporter dye molecules in an optimized buffer. Transfer into a qPCR plate's wells.
Add heat-treated bacteriophages. Heat treatment releases bacteriophage DNA from bacteriophage particles. Seal the plate, preventing mixture evaporation. Load the plate into the qPCR system, setting appropriate cycling conditions.
Heating to high temperatures denatures the double-stranded DNA into single strands. It also activates the Taq DNA polymerase. Annealing causes sequence-specific primers to bind to the complementary bacteriophage DNA strands. Extension allows activated Taq DNA polymerase to add dNTPs to the 3' end of the primer, extending the growing DNA strand in the 5' to 3' direction.
The dye molecules intercalate into the newly synthesized double-stranded DNA, causing increased fluorescence intensity. Each PCR cycle doubles the target DNA amount, increasing the fluorescence intensity.
Determine the threshold cycle, Ct, value, at which measured fluorescence exceeds the background level.
The Ct value is inversely related to the starting DNA amount, with each bacteriophage genome equivalent to one bacteriophage particle, facilitating bacteriophage quantification from the bacteriophage DNA standard curve.
Post-amplification, perform melt curve analysis, gradually increasing the reaction temperature. At a specific melting temperature, half the DNA separates into single strands, releasing dye molecules and causing sudden fluorescence decrease.
A distinct melting temperature, unique for the qPCR product, validates the product specificity.
Prepare the qPCR premix for 10 biopanned phage samples of unknown concentration and 7 samples of standard concentrations in triplicate, resulting in a premix for 51 samples containing 255 microliters of qPCR Master Mix, 51 microliters of primers, and 102 microliters of water.
Aliquot 8 microliters of the prepared PCR mix into 51 wells of a 96-well qPCR plate. Next, add two microliters of heat-treated T7 phage samples to each of these wells, dispensing it below the surface of the qPCR premix. Using adhesive film, seal the plate.
Wrap the plate in aluminum foil to minimize fluorescence photobleaching, and then, proceed to run the plate on the qPCR equipment. Set up the cycling conditions and melt curve settings as outlined in the text protocol.
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