12: OUTBREAK
- Page ID
- 157072
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Interpret simulated clinical or laboratory data to infer possible bacterial causes of food borne illness based on DNA analysis.
- Explain how DNA analysis is used to identify a causative agent.
BACKGROUND
According to the Centers for Disease Control and Prevention (CDC), approximately 48 million Americans get sick, 128,000 are hospitalized and 3,000 die each year from food poisoning. In 2000, 406 cases of the same food borne illness were reported to the CDC across ten states, mostly on the West Coast. Epidemiologists working on the case identified the mode of transmission to be the oral fecal route. A bacterial organism was isolated from stool samples taken from 98% of patients with similar signs and symptoms. There are several possible Enterobacteriaceae organisms that could be causing the illness. Enterobacteriaceae are Gram negative, non-endospore forming, facultative anaerobes. Enterobacteriaceae pathogens commonly associated with food borne outbreaks include Shigella, Salmonella and E. Coli. The sign and symptoms patients experience from these various bacterial pathogens can be very similar, yet effective treatments can be very different.
Illness from these pathogens range from mild diarrhea to severe dysentery depending on the toxins the bacteria produce. Some Shigella strains contain Shiga toxins, potent cytotoxins that can cause severe disease and can ultimately result in death. Typically, Shigellosis is self-limiting, (recovery occurs without medication,) but the patient should be monitored and kept hydrated. Enteroinvawsive E. coli (EIEC) is very similar to Shigella species and produces toxins that very similar signs and symptoms and requires medical treatment. Infections by E. Coli O157:H7 can also initially present very similar to Shigella but progress to be extremely severe leading to death if not properly treated. Salmonella is the name of a group of bacteria that causes salmonellosis. Salmonellosis treatment primarily focuses on managing symptoms like diarrhea and preventing dehydration through fluid and electrolyte replacement. Antibiotics are generally reserved for severe cases or those at high risk of complications. What toxins a bacterial organism produces depends on its genome (sum of all genetic material within a cell including the chromosome and plasmids present). For diagnostic and treatment purposes it’s often important to quickly determine what bacteria and specific strain is infecting the patient. In the past the only way to determine what pathogen was causing a patients disease was the signs, symptoms presented along with metabolic testing. Metabolic testing can take several days. With increasing frequency, hospitals and clinical care labs analyze the pathogens DNA to quickly identify which microbe is infecting the patient. The DNA analysis techniques used to identify the bacterial pathogen include Polymerase Chain Reaction and gel electrophoresis.
Polymerase Chain Reaction (PCR) is a technique used to make millions of copies of a specific DNA segment. It works by cycling through heating and cooling steps that break apart DNA strands, attach short primers to the target sequence, and build new strands using an enzyme called DNA polymerase. PCR is fast, highly sensitive, and allows scientists to detect and study even tiny amounts of DNA—making it especially useful in diagnosing infections, solving crimes, and studying genes.
Gel electrophoresis is a technique that uses electric current to separate a mixture of organic molecules based on their sizes and chemistry. As DNA molecules have negatively charged phosphate groups, the molecules will migrate through the solid gel towards the positive electrode. Commonly made from agarose or polyacrylamide, the solid gel matrix has a mixture of small holes and large holes. As small molecules migrate faster through the gel, the molecules from different samples can be analyzed for size comparison.
MATERIALS (Per Group of 4)
1 P-20 Micropipettes
1 Micropipettes tips
1 Beaker for waste
1 1% Agarose TAE gel
1 Gel electrophoresis system
1 Microfuge tube rack
5 1X TAE DNA samples
1 1X TAE Size marker
METHODS/PROCEDURES
HOW TO USE MICROPIPETTES
Adjusting Volumes
The instrument pictured at the top right is a typical P20 micropipette (a precision tool designed to measure and transfer very small liquid volumes). The “P20” name means it can hold up to 20.0 microliters (µL) of liquid. This micropipette is accurate for volumes between 2.0 µL and 20.0 µL. It should not be used for amounts smaller or larger than this range.
Micropipette volumes are adjusted using a three-digit dial located on the handle. Many micropipettes use a color-coded digit to indicate the decimal point. For example, in a P20 pipette, a setting of black 0, black 2, red 0 indicates 2.0 µL.
Loading the Micropipette
- Attach a disposable tip
Always use a clean, disposable plastic tip. Push the micropipette’s shaft straight down into a tip (located in the tip box) until it clicks firmly into place. - Set your volume
Dial in the volume needed using the adjustment knob. Make sure the setting is within the P20’s valid range. - Grip the pipette correctly
Hold the micropipette like a pencil, wrapping your fingers beneath the curved finger rest, with your thumb on the plunger button. - Understand the two “stops”
- First stop: Pressing the plunger gently until you feel the first stop is used to draw up liquid.
- Second stop: Pressing beyond the first stop to the second stop is used to eject all the liquid from the tip.
Transferring Liquids
- Aspirate (draw up liquid)
- Press the plunger down to the first stop and hold.
- Submerge the tip in the liquid sample.
- Slowly release the plunger to draw liquid into the tip.
- Check for smooth intake with no air bubbles.
- Dispense (release liquid)
- Move the pipette to the receiving tube or surface.
- Press the plunger to the first stop to begin dispensing the liquid.
- Continue pressing to the second stop to ensure all the liquid is released.
- Eject the used tip
- Always change the tip between samples to avoid contamination.
- Press the tip ejector button to discard the used tip into a designated waste container.
1.3 Micropipetting is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Orange County Biotechnology Education Collaborative (ASCCC Open Educational Resources Initiative).
Display Window
1.3 Micropipetting is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Orange County Biotechnology Education Collaborative (ASCCC Open Educational Resources Initiative).
METHODS
1. Set the P20 micropipet to 10.0 µL. The dial should read 100, top to bottom
2. Place the 1% agarose gel into the electrophoresis chamber. Pour enough 1X TAE buffer to just cover the gel (entire gel needs to be completely submerged).
3. Load 10.0 µL of each DNA sample into individual wells of the gel in the order indicated in the chart below changing tips for each sample.
4. Once all samples have been loaded, place the cover on the gel tank, matching colored electrodes.
5. Plug the gel box into the power supply and set the voltage to 120V.
6. Run the gel for 15-30 minutes or until the samples are separated into distinct bands.
7. Remove the gel from the electrophoresis box and view it on the light box
Example
Mnolf, CC BY-SA 3.0 <http://creativecommons.org/licenses/by-sa/3.0/>, via Wikimedia Commons
OUTBREAK
NAME ______________________
EXPECTATIONS
If E. coli O157:H7 contains an extra plasmid that regular E. coli does not, would you expect their DNA band patterns on the gel to be the same or different?
Describe what you would expect to see on the gel and explain why.
Where do you expect plasmid DNA bands to appear on the gel compared to chromosomal DNA bands, Explain why?
RESULTS
Draw the results of the gel electrophoresis below
CONCLUSIONS
1. Based on the results above, what pathogen caused the outbreak? ________________________________
2. Explain how you made your determination.
3. Explain one advantage and one disadvantage of using DNA testing instead of metabolic testing to identify
bacteria.
4. Explain one advantage and one disadvantage of using metabolic testing to identify bacteria instead of DNA
testing.
5. Why do you think hospitals are switching to DNA-based identification methods instead of relying only on
symptoms or metabolic tests?