The overall goal of this procedure is to obtain genotypes of Staphylococcus aureus, and to link them with disease in hosts. This method can help answer key questions in the field of bovine mastitis, to protect contagiousness and passaginicity of Staphylococcus aureus, isolate it from cows with intramammary infection. Demonstrating the procedure will be Ivana Ivanovic, a technician from my laboratory.

To begin, spread 10 microliters of Staphylococcus aureus stock culture, or one colony grown on selective medium, on Columbia Agar plates containing 5%sheep blood, or BA.Incubate the culture aerobically at 37 degrees Celsius for 18 to 24 hours. Use a sterile plastic loop or wooden toothpick to collect one bacterial colony from a BA agar plate, and re-suspend it in 100 microliters of TEL in a 1.5 milliliter screw cap tube. Incubate the samples at 95 degrees Celsius for 10 minutes and immediately place them on wet ice.

Then, dilute the samples one to 100 in water suitable for PCR. Prepare a master mix by adding to a tube the reagents listed here. Then, distribute it to PCR tubes and add the sample DNA.

Include a positive control, DNA generating a known genotype, and water as a negative control. Use a standard PCR cycler to run the program discussed in the text protocol. After approximately six and three quarters hours of cycling, stop the machine and take out the tubes.

To carry out electrophoresis, use a commercial miniaturized electrophoresis kit for DNA, together with a Bioanalyzer, connected to a PC and the installed software. Set up the chip priming station and adjust the syringe clip according to the manufacturer's instructions. Next, put a new DNA chip in the chip priming station.

Then, after equilibrating the gel dye mix to room temperature, pipette nine microliters of the gel dye into the well marked G.Make sure that the plunger is positioned at one milliliter, and close the chip priming station. Press the plunger until it is held by the syringe clip, then wait for exactly 30 seconds and release the syringe clip. After waiting for five seconds, slowly pull back the plunger to the one milliliter position.

Open the chip priming station, and pipette nine microliters of gel dye into both wells marked G.To load the marker, pipette five microliters of marker into all 12 sample wells, and into the well marked with the ladder sign, ensuring that all 13 wells are loaded. Pipette one microliter of DNA ladder into the ladder well. Then, pipette one microliter of sample into the sample wells and one microliter of deionized water into the unused wells.

Place the chip horizontally in the adaptor and vortex for one minute at 2, 400 RPM. Within five minutes of vortexing the chip, start the Bioanalyzer and computer, and open the software. Open the lid of the Bioanalyzer and place the chip into the chip holder.

Then, carefully close the lid. In the instrument context of the software, select electrophoresis, double stranded DNA, DNA 7500. Accept the current file prefix of the software, or modify it.

Click the start button present in the software, then enter the sample names. When the chip run is complete, the end of run message will appear in the Bioanalyzer software. Remove the chip and clean the Bioanalyzer electrodes according to the manufacturer's instructions.

Finally, infer the genotype from the electrophoresis profile according to the text protocol. As seen in this Bioanalyzer pseudo-gel of 12 RS-PCR products and their inferred genotypes, variation in just one band is regarded as a genotypic variant. Each lane is characterized by a pattern of three or more PCR bands, and a marker band in front, and a marker band at the end.

This electrophoretic profile for sample 161 measures the intensity of the bands as individual peaks of fluorescent units. Another electrophoretic profile, for sample 211, reports a peak for each band as the number of base pairs. Peak reading in fluorescent units is required for evaluating the relevant peaks of a profile in the Mahal software.

And reading in base pairs is for inferring the genotype. The relevant peaks identified for sample 211 are shown here, inserted in the Mahal software. The calculated squared Mahalanobis distance, or D2M, between the queried and indicated genotypic profile, then identifies a genotype that is minimal and not significantly different than the one queried.

In this case, GTB. Once mastered, the complete analysis of 96 samples requires one night for PCR and one day for electrophoresis and evaluation. Pipetting for sample dilution and setting up the PCR can be massively simplified if a robot is applied.

While attempting this procedure, it's important to remember to include positive and negative PCR controls, such as DNA for a particular genotype, and water respectively. Follow this procedure. Other methods like spa typing, or analysis of virulence genes, can be performed in order to further characterize the strains of Staphylococcus aureus.

After its development, this technique paved the way for researchers in the field of bovine mastitis to explore highly sensitive and specific qPCR assays for clinically relevant genotypes of Staphylococcus aureus. Notably, genotype B.After watching this video, you should have a good understanding of how to establish and drum RS-PCR, particularly to infer the Staphylococcus genotypes. Don't forget that working with Staphylococcus aureus can be hazardous, and good laboratory practices should always be applied while performing this procedure.