The overall goal of this procedure is to determine the SCC MEC type of methicillin resistant staphylococcus aureus or MRSA isolates using a multiplex PCR assay. This is accomplished by first isolating DNA from the strains, using a rapid and simple extraction technique involving heating of the bacterial cells in water. The second step is to perform a multiplex PCR assay in order to amplify targets specific to each of the SEC ME types.
Next, the PCR products are separated by AROS gel electrophoresis. The final step is to stain the gel for visualization of the DNA and data analysis. Ultimately, the unique combination of specific PCR products obtained with this multiplex PCR assay is used to determine the SE cmec types of the MRS A isolates The main advantage of this technique over existing methods such as traditional S CEC typing schemes, which target individual CCR and MEC gene complexes and require multiple individual PCR tubes, is that this process greatly simplifies a workload and can be done in a single PCR tube.
These procedures should be conducted in a certified biosafety level two laboratory. Appropriate personal protective equipment should be used at all times on the day before PCR is to be done. Select a single colony of MRSA from a fresh overnight plate with a sterile culture stick and prepare a heavy streak of the bacteria on a triptych soy agri plate.
Multiple samples can be streaked onto a single plate incubate overnight at 37 degrees Celsius. Add 75 microliters of sterile distilled water to a 1.5 milliliter micro centrifuge tube. Using a sterile culture stick.
Pick up a small amount of bacteria from the heavy overnight streak. Swirl the bacteria in the sterile water to prepare a turid solution. Repeat this process for all samples.
Incubate the tubes in a dry heat block set at 95 degrees Celsius for 10 minutes to ly the bacteria and release the DNA. Remove the samples from the heat block and lets stand at room temperature for five minutes. Centrifuge the samples at 13, 000 RPM for one minute.
The resulting sample has a cellular debris pellet and clear supernatant containing the DNA. To prepare for PCR, remove the PCR reagents including 10 XPCR buffer, 50 millimolar magnesium chloride, DTPs and primers from the minus 20 degrees Celsius freezer and allowed to thaw at room temperature on the clean PCR bench where no bacteria or DNA is permitted. 0.2 milliliter PCR tubes can be prepared and labeled at this time.
Once thawed vortex briefly to mix all reagents, then combine as tabular in the text protocol. It is important to use platinum tech DNA polymerase as it has been found to influence the success of the reaction. After vortexing the master mix Eloqua into the individual PCR tubes, close all PCR tubes and move to the general workbench.
Add the template to the PCR tubes by pipetting two microliters of the clear supernatant from the previously prepared bacterial samples. Move to the dedicated amplification location before placing the tubes into the thermal cycler and running the reaction as listed in the text protocol. Working in a dedicated gel electrophoresis location at 2.5 grams of agarose to 100 milliliters of 0.5 XTBE buffer in a glass erlenmeyer flask.
Cover the flask with plastic wrap and boil the solution in a microwave until fully dissolved. A 2.5%gel will boil over quickly. Therefore stop the microwave and swirl the solution regularly.
Cool the agros until comfortable to touch by swirling under cold water or by resting on the bench for five minutes. Pour the agros into the casting tray after removing the tubes from the PCR thermal cycler. Prepare the samples by adding two microliters of six XDNA loading dye to each sample.
Load five to 10 microliters of sample per well on the gel. A one KB plus or 100 base pair DNA ladder is appropriate for the expected band sizes. A th dium bromide can be added to the gel prior to pouring it or alternatively, the gel can be stained in an atherium bromide bath prior to being visualized.
Place the finished gel in a solution of 0.5 micrograms per milliliter, atherium bromide and distilled water and mix gently on a shaker for 10 minutes. Transfer the gel to a clean container and detain in distilled water for 10 additional minutes. Shown here is a representative gel demonstrating the expected PCR products for all representative strains of SEC MEC types one through six and eight as well as SEC MEK four subtypes A through F.For a typical SEC MEK one strain two bands are expected.
The type one specific band at 613 base pairs as well as the mec, a control band at 147 base pairs. Three representative SEC MEK two strains are shown including the prototypical type two of strain N three 15 and the less prevalent Type two A and type two B To classify a strain as SEC mek two two bands should be present including the type two locus target at 128 base pairs and the Meca control two representative SEC MEK three strains are shown on the gel including the prototypical SEC MEK three of strain 85 2 0 8 2 and the less type three A of strain. JCSC two 90 SSC MEC three is identified based on the presence of two PCR products, including the type three specific target at 257 base pairs and the Mecca control target.
The dominant SEC MEC three will also be positive for the mercury target at 280 base pairs, which is carried on an adjacent SEC element and serves to distinguish it from the less common SEC MEC three A five. Representative SEC MEC four elements are shown including types four A, four B, four C, four D, four E, and four F.Unlike with SC MEK one through three, there is no single target present in all of the SCC MEK four subtypes apart from meck A.The representative SCC MEK four E strain is identified based on the presence of the Type four E band as well as the CCR AB four product, which is present on an adjacent accessory element and the type four C product which is present because SEC MEK four E shares the same J one region as SE Cmk four CSCC MEK four F is similar to SEC MEK four E in that it shares the same J three region and is consequently positive for the four E target as well as as positive for the CCR AB four target due to an adjacent accessory element. S Cmk four F can however be distinguished from S CMK four E in that it lacks the type four C target.
A typical SE CMK five would've. Two PCR products present on the gel, the type five specific band at 325 base pairs and the ME a control at 147 base pairs. Finally, while the goal of this assay was not to provide definitive identification of either SEC MEK six or eight representative strains were included for both since a specific PCR pattern provides suggestive evidence for the presence of these types.
In addition to the Meck A target, the representative SEC MEK six would be expected to produce a positive CR AB four product at 106 base pairs as this is its main CCR complex type. The representative SEC MEC eight would also be positive for MEC A at 147 base pairs and C-C-R-A-B four at 106 base pairs and would be distinguished from SEC MEK six on the basis of a positive type two specific product which results from it having the same J two region SEC MEK two. While attempting this procedure, it is important to remember that due to the sophisticated nature of the assay and the large number of primers present in the reaction there is the potential for difficulties.
While adapting this to individual laboratories, paying close attention to certain key issues such as DNA template preparation and PCR Reagent brand will greatly facilitate this goal.
