Pulsed-Field Gel Electrophoresis is a technique used to identify particular strains of bacteria most notably E. coli O157:H7 through DNA “fingerprinting.”
What happens in PFGE?
The DNA of the unknown strain of E. coli bacteria is first isolated. Next, the DNA is cut up into pieces by enzymes called restriction endonucleases. Restriction endonucleases are vital to PFGE because there are many different types of these enzymes that each cut DNA at a specific base sequence. Each strain of E. coli has a slightly different strand of DNA, each with unique restriction sites where the DNA can be cut by the restriction endonucleases. So when the unknown strain of DNA is chopped with at least two different restriction endonucleases, the process will generate segments of DNA that may be unique in size or number of segments.
These segments of DNA are then placed in an agarose gel (a gel made from algae or seaweed) which is connected to a voltage source. Unlike basic gel electrophoresis that uses a continuous current, PFGE uses an alternating, pulsed current. The reason for the pulsed current is that the segments of DNA produced by the restriction endonucleases are too large to be clearly separated by continuous current, but pulsing allows the large fragments to separate. PFGE also differs from typical gel electrophoresis because the gel must run much longer, up to 24 hours, to compensate for the large fragment sizes.
After the gel has finished running, it is treated with a visualizing agent and a photograph is produced for easy comparison. The final product looks like this:
Each lane represents one strain of E. coli DNA or a known standard by which to compare the unknown strain’s DNA.
PFGE and Outbreaks
PFGE is so powerful because it easily allows scientists to visually compare strains of E. coli and determine their relatedness. For example, if the hamburger of a specific restaurant is the suspected source of an E. coli outbreak, scientists will take samples from the ground beef and stools of sickened individuals. If E. coli is found in each sample, the DNA will be processed using PFGE. After PFGE is completed, scientists compare the banding patterns in the gel from the ground beef E. coli to that in the sick individuals. If the banding patterns match up exactly, it is highly likely that the same strain was found in both the restaurant and the sick individuals, possibly implicating the restaurant as the source of an outbreak.
PulseNet
The CDC collects data obtained by laboratories using PFGE on the different strains of E. coli O157:H7. This national computer database is available to investigators and researchers, allowing them to compare E. coli O157:H7 strains nationwide and uncover multi-state E. coli outbreaks.
Multi-Locus Variable-Number Tandem Repeat Analysis
Although PFGE tests can help investigators find the source of an E. coli infection, individual tests are time consuming and can only be performed by specially trained researchers. A new technique to identify different strains of E. coli called multi-locus variable-number tandem repeat analysis (MLVA) is now being investigated by the CDC. MLVA has been found to be easier to reproduce and able to more easily distinguish between closely related strains, potentially making it a better candidate to map sources of E. coli outbreaks than PFGE.
Sources:
1. Johnson JM, Weagant SD, Jinneman KC, Bryant JL. 1995. Use of Pulsed-Field Gel Electrophoresis for Epidemiological Study of Esherichia coli O157:H7 during a Food-Borne Outbreak. Applied and Environmental Microbiology, 61:7: 2806-2808.
2. Centers for Disease Control. PulseNet. 2006. Online at http://www.cdc.gov/pulsenet/
3. Cooley M, Carychao D, Crawford-Miksza L, Jay MT, Myers C, et al. 2007. Incidence and Tracking of Escherichia coli O157:H7 in a Major Produce Production Region in California. PLoS ONE 2 11: e1159. doi:10.1371/journal.pone.0001159
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