The overall goal of the following experiment is to develop A-Q-P-C-R assay that can be used for selective detection of live e coli oh 1 57 H seven cells. Open reading frame Z 32 76 has been identified as a unique genetic marker that can be used in a Q PCR R assay, which is sensitive and specific for e coli oh 1 57 H seven following preparation of live and dead cell mixtures. They are incubated with propidium mono acid or PMA agitation allows PMA to get inside the dead cells, but not inside the live cells.
Next, the DNA is extracted and QPCR is performed on the treated and untreated live and dead cells Results are obtained that show the PMA treatment efficiently removes DNA from dead cells and essentially does not affect the amplification of DNA from live cells leading to the specific detection of live e coli oh 1 5 7 H seven cells. The main advantage of this technique, our existing methods, like the available QPCR assay for detection of e coli oh 1 5 7 H seven, is that this protocol can be used for selective detection of live e coli oh one seven H seven cells. This method can help answer key questions in the food boy pathogen detection field, such as the overestimation on DNA of live cells in samples.
The implications of this technique may extend towards detection of other pathogens and using Z 32 76 as a unique genetic marker for e coli, O 1 57. H seven provides the assay with high sensitivity and specificity and the potential for multiplex assays. Grow 10 milliliters of e coli oh 1 5 7 H seven at 37 degrees Celsius to mid exponential phase and divide the culture into two aliquots.
Boil one aliquot of cells for 10 minutes in a water bath for dead cells, and leave the other aliquot for live cells. Verify there are no live cells from the heat killed aliquot by plating the cells on LB APLs and incubating it 37 degrees Celsius overnight. Meanwhile, take two milliliters of the live and heat kilt cells and adjust the concentration to 8 million CFUs per milliliter with LB medium create four sets of live cell dilution ranging from eight CFUs per milliliter to 8 million CFUs per milliliter for the first two sets of cell dilution label.
One as live cells treated with PMA and the other as the untreated live control sample to the third and fourth sets of cell dilu. Add 8 million dead cells to each cell dilution to make live and dead cell mixtures. The third set of dilu will be treated with PMA and the fourth set of dilu will serve as the untreated mixture control at aliquot 400 microliters of the live cells, dead cells, and mixture of live and dead cells.
In three separate micro tubes, add two microliters of a 10 millimolar stock solution of PMA to each cell, aliquot to a final concentration of 50 micromolar. Incubate the samples at ambient temperature for five minutes in the dark during the incubation. Shake each tube gently a few times, three seconds per time.
Next, add 100 microliters of the samples into a 96 well plate seal the plate with an optical film and put the plate on ice. Set the plate 20 centimeters from a 650 watt halogen light source and expose the plate to the light for two minutes. Then centrifuge the plate at 2, 500 cheese for 10 minutes following centrifugation, gently discard the snat and carefully drain the plate on a piece of absorbing paper.
Re suspend the cell pellets in 50 microliters of DNA extraction solution by pipetting up and down 20 times with a multi-channel Pipet man seal the plate with a film and boil for 10 minutes in a water bath before centrifusion at 2, 500 Gs for two minutes. The supernatant in the plate is the DNA from cells treated with PMA and is ready for QPCR. Prepare the QPCR reaction mixtures by combining two x real-time PCR master.
Mix forward primer and reverse primer and probe. See the text protocol for details of primer and probe design. Then transfer the QPCR reaction mixture to individual wells of a new 96 well plate.
For each of the treated and untreated live and dead cells, add five microliters of sample DNA and an appropriate volume of water to reach a final volume of 50 microliters. For the non template control, use five microliters of water to replace the DNA sample. Next, set the QPCR conditions as 95 degrees Celsius for 10 minutes for activation of TAC man, followed by 40 cycles of 95 degrees Celsius for 10 seconds for denaturation and 60 degrees Celsius.
For one minute of an kneeling extension, proceed to perform QPCR on the treated and untreated live and dead cells. The effects of PMA mediated inhibition of amplification of DNA from dead cells on this QPCR assay are shown here by using DNA derived from live or dead cell dilutions. The results showed that the curves generated from the live cells treated with PMA and without PMA seemed linear and nearly identical to one another.
Slight threshold cycle or CT value differences were shown between the live cells treated with PMA and without PMA suggesting that PMA treatment had little effect on amplification of DNA of the live cells in the QPCR. Conversely, a 15 CT value difference was shown between the dead cells treated with PMA and without PMA demonstrating that PMA treatment efficiently suppressed DNA amplification from the dead cells. Two sets of live cell dilution were treated with PMA or without PMA to detect live cells from live dead cell mixtures.
The QPCR results demonstrated a parallel inverse progressive trend of CT values as related to the numbers of treated live cells to the untreated samples. The treated live cells yielded somewhat higher CT values than those of untreated live cells. A similar inverse progressive trend in CT values was observed with the real number of live cells in the live dead cell mixtures treated with PMA.
In addition, this downward trend of CT values was observed with the number of live cells in the mixtures despite the presence of a million dead cells. These results showed that the CT values of the live dead cell mixtures represented the DNA from the live cells alone, and that DNA amplification from the dead cells was efficiently suppressed by PMA treatment Once mastered. This technique can be done in two hours if it is performed very well While attempting.
This procedure's important to remember to have a good light exposure for the PM treated samples to ensure the PME treatment is thorough and at the same time to protect the live cell in the samples Following this procedure. Other methods like QPCR assays can be performed as a regular real-time PCR assay.
