A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.
This method could help answer key problems in the virology and microbiology fields related to enumeration of bacteriophages from complex samples and bacteriophage based diagnostics and therapeutics. The main advantage of this technique is that it enables time efficient and accurate quantification of a large number of samples from phage display biopanning experiments not feasible with traditional assays. Demonstrating the procedure will be Rashmi Mohanty and Jasmim Leal, graduate students from my laboratory.
After designing the primers and creating a stock concentration, obtain reference T7 phage DNA at a concentration of 0.1 micrograms per microliter. Using ultrapure water, serially dilute this DNA tenfold, from one million femtograms per microliter to one femtograms per microliter in 0.2 milliliter PCR tubes. Prepare 20 microliters of each concentration, making sure to change pipette tips and vortex the tubes between each stage of the dilution.
Then convert the T7 phage reference DNA concentration from femtograms per microliter to genome copies per microliter. First, pipette 200 microliters of the sample T7 phage clone into a 1.5 milliliter micro centrifuge tube for each of the 10 desired samples. Add two microliters of DNase one to each tube.
Cap the tubes and incubate at 37 degrees Celsius for 10 minutes in a heated dry bath. Then incubate at 100 degrees celsius for 15 minutes in a heated dry bath. Centrifuge the tubes containing the heat treated phages at 18, 534 times g for 10 seconds.
Place the centrifuged tubes on a vortex mixer set at touch activation mode for 10 seconds to mix the phages. Spin again at 18, 534 times g for 10 seconds. After this, leave the samples on the laboratory bench about one hour to cool them to room temperature.
Prepare the qPCR premix for 10 biopanned phage samples of unknown concentration and seven 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 eight 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 photo bleaching 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. After the qPCR is run, the amplification plot, melt curve plot, and multicomponent plot are analyzed from the raw data. The amplification plot indicates whether the PCR amplification of each sample is successful or not.
The multicomponent plot represents the amplification and decay of the fluorescent signal throughout the amplification cycles. While the melt curve plot is used to validate the specificity of the primers, since each PCR product has one unique melting temperature. The standard curve is then generated from the reference DNA.
Here the amplification efficiency is calculated to be 92.4%Representative data from a qPCR study is then compared to the same phage that were quantified by a double layer plaque assay at the tested range of 10 to 10 million genome copies per microliter. As seen here, the number of phages in the qPCR quantitation is higher than in the plaque assay. The repeatability of the qPCR method as represented by the coefficient of variance is seen to have values ranging from 2.85 to 27.2%While attempting this procedure, it's important to remember that there are numerous considerations in the development and use of qPCR to accurately quantify phages, including careful treatment and preparation of samples and calibration of relevant equipment.
Following this method, other techniques can be performed in order to enumerate phage particles in a variety of applications included in vivo biopanning, phage library DNA preparation and next generation sequencing and phage detection in complex environmental samples. Don't forget that phages are considered biological hazards in the laboratory and precautions such as personal protective equipment, including gloves and lab coats, should always be worn while performing the procedure.
