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Diagnosing H1N1 Using RT-PCR
Materials Needed per Student / Student Group:
Lab Handout
OSHA BSL Fact Sheet
Excel File of RT-PCR Data
Laptop with internet connection and Excel Program
Learning Objectives:
1 – Gather information on H1N1 from a reliable source.
2 – Learn safe practice handling procedures for researching various pathogen types.
3 – Better understand how various types of PCR can be used in diagnosis in the public
health field.
4 – Critically analyze results from an RT-PCR data set.
5 – Learn about reporting of various diseases and how that affects national statistics.
Section 1: What is H1N1 Influenza?
Open your laptop computers and open the browser. In the URL box, type in:
www.cdc.gov
In the box at the upper right side of the page, type in “H1N1” and hit enter.
Click on the first link that appears – “CDC H1N1 Flu | H1N1 Flu and You”
1. When was this site last updated? (Shown in red at the top of the page) _________
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2. What does this indicate about the CDC’s current level of activity regarding
monitoring H1N1 and reporting this information? ____________________________
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3. Summarize the information found in the first section “Flu Activity”. __________
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4. Summarize the information found in the second section “Vaccination”. ________
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Now scroll down and find the box on the right side of the page entitled “Current or New
Information”. Click on the first link, “CDC Response: A Year in Review”.
5. Before H1N1 became a known pandemic, the CDC health officials were focusing
pandemic mitigation efforts on a different strain of flu. What was this strain? _____
_____________________________ What animal acted as a reservoir for this strain?
________________________________________________________________________
6. When was H1N1 first identified in the United States? _______________________
7. From where did the genes for this strain of H1N1 come? _____________________
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Is this classified as Influenza A, Influenza B, or Influenza C? ___________________
8. How is it possible for these genes from all over the world to be combined in one
viral strain? _____________________________________________________________
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9. To which drugs is this virus resistant? ____________________________________
To which drugs is it susceptible? ___________________________________________
10. By April 21, 2009, the CDC started trying to find a virus that would make a
good vaccine. What was their first step at vaccine preparation? _________________
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11. How do they grow these viruses in the CDC labs? _________________________
12. April 27, the CDC increased the alert from phase 3 to phase 4. What caused this
move? _________________________________________________________________
13. April 29, the CDC increased the alert from phase 4 to phase 5. What caused this
move? _________________________________________________________________
14. In addition to the usual symptoms of the flu, what other symptoms do H1N1
infected patients have? ___________________________________________________
Further analysis:
15. Go to Wikipedia and type in the name of the drugs H1N1 can resist. Write their
names below with a short description of their mode of action.
________________________________________________________________
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16. Then find the drugs, which are successful in treating H1N1, type those into
Wikipedia, and write the name of the drug below with a short description of their
mode of action.
________________________________________________________________
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Section 2: What is a BSL Lab?
H1N1 is clearly a human pathogen, so precautions must be taken when handling, working
with, or growing this virus. Read the attached worksheet from OSHA (Occupational
Safety and Health Administration) on BSL practices and answer the following questions.
1. When are BSL1 labs used? _____________________________________________
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2. Summarize the recommendations when working in a BSL1 lab below. _________
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3. When are BSL2 labs used? _____________________________________________
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4. Summarize the recommendations when working in a BSL2 lab below. _________
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5. When are BSL3 labs used? _____________________________________________
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6. Summarize the recommendations when working in a BSL3 lab below. _________
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7. When are BSL4 labs used? _____________________________________________
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8. Summarize the recommendations when working in a BSL4 lab below. _________
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9. Based on what you now know of BSL labs, which lab do you think would be
required for working with H1N1? Why? ___________________________________
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Section 3: What is RT-PCR?
Remember that PCR is used to amplify (copy) target DNA in a test tube in a short period
of time (just a few hours). The steps of PCR include denaturation, at 95oC, annealing,
usually around 60oC, and extension at 72oC. Also remember that we analyzed our PCR
data by running the amplified DNA products, also known as amplicons, in a gel and
staining the gel for our DNA product. This additional step, of running and staining the
amplicons in a gel, takes another few hours, doubling the time to detecting your product.
In addition, you can only gather data on the last cycle, and the amount of amplicon
produced is not entirely quantifiable.
Real-Time PCR, on the other hand, allows you to monitor amplification as it is happening
in the PCR reaction wells. Watch the video found at the following link and answer the
questions below.
http://www.youtube.com/watch?v=N6NhyMMMvyA
1. Other than the thermal cycling system (PCR machine) and the reaction mix
itself, what one added component is needed in an RT-PCR machine? ____________
________________________________________________________________________
2. Is the RT-PCR thermal cycling reaction different from regular PCR steps? _____
If so, how? ______________________________________________________________
3. SYBR Green is added to the RT-PCR mix. It does not fluoresce in solution, so no
signal is picked up by the computer. When does it fluoresce? __________________
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4. When does the computer gather the data from SYBR Green fluorescence?
________________________________________________________________________
The video you just watched shows the more traditional methods of using RT-PCR.
However, today’s RT-PCR is more specific and more sensitive to the target DNA of the
reaction. Additional components, known as hydrolysis probes, are added to the
traditional RT-PCR mix to increase specificity and sensitivity. Remember that nucleic
acid probes are short strands of complementary sequences to your target DNA. They will
bind via hydrogen bonds to your target DNA during the reaction. Watch the video found
at the following URL and answer the questions below.
http://www.youtube.com/watch?v=8jBSAca9XNU
1. The nucleic acid probe used in this modified version of RT-PCR has two
chemical groups attached on either side. What are these two chemical groups and
what is their function? ____________________________________________________
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2. During the reaction, the reporter dye is cleaved from the probe. What enzyme is
responsible for this activity? _______________________________________________
3. What happens as a result of this cleavage? ________________________________
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4. What are the two disadvantages of RT-PCR mentioned in the video? __________
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5. In addition to the advantage of increased specificity, the video also mentions
hydrolysis probes can be used in multiplexing in RT-PCR. What is multiplexing
and what are the advantages of it? __________________________________________
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6. What are the three applications of multiplex RT-PCR mentioned in the video?
________________________________________________________________________
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Further discussion:
Real-Time PCR can be modified as well to incorporate additional steps, namely reverse
transcription. Reverse transcriptase RT-PCR is used to diagnose H1N1 infections in
patients.
1. What does reverse transcriptase do? _____________________________________
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2. Knowing that influenza is an RNA virus, why would this additional step be
necessary in diagnosing infections? _________________________________________
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Section 4: Analysis of RT-PCR Results
Find the RT-PCR data file on your computer and open this file. RT-PCR data can show
you the change in reaction fluorescence (Rn), which is the readout of fluorescence data
for each reaction. However, this is a lot of information to filter through. Partly, this is
because the reporter dye’s fluorescence is not quenched 100%, and there is always a
background of fluorescence during the first 10 – 15 cycles. However, if the template is
present, eventually the fluorescence begins to increase exponentially and will rise quickly
above the background fluorescence. Based on how strong the background fluorescence
is, a threshold is set, so that when the fluorescence increases above this threshold, a
positive reaction is recorded. The reaction cycle when fluorescence rises above threshold
is given the name “Ct”, for “cycle threshold”. Ideally, a Ct value is trusted if it is
between 25 and 35, meaning the amplification fluorescence rose above background
fluorescence between the 25th and the 35th cycle. If the Ct value is above 40, it is
considered a negative reaction.
In addition to testing for potential pathogens, there are also positive controls and negative
controls run in every reaction. The positive control is to ensure the reaction is running
properly. A common positive control to run in reverse transcriptase RT-PCR reactions is
RNase P. This assays for the presence of the RNA transcript of the gene RNase P, a gene
expressed in most cells throughout the body. Every sample taken from a human patient
should contain a large amount of this RNA molecule.
The negative control includes all the reaction ingredients (buffer, enzyme, primers, probe,
dNTPs), but doesn’t include the patient sample; instead, we just add water. There should
be no reaction in these wells. If there is, you may have contamination.
Take a look at the RT-PCR data file and answer the questions below. Please note: “InfA”
tests for Influenza A, “SW H1” tests for swine flu viruses that contain the H1 gene (this
includes H1N1) and SARS. “NTC” stands for “No Template Control” and is the
negative control for each reaction.
1. Of the three patients, which one(s) is/are infected with influenza A? ___________
2. Of the three patients, which one(s) is/are infected with H1N1? _______________
3. Of the three patients, which one(s) is/are infected with SARS? _______________
4. Were there any reactions where the positive control did not turn out properly?
____________ If so, why might this occur? __________________________________
What could you conclude about your reactions if the positive control did not turn
out well? ______________________________________________________________
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5. In Patient #1, one of the RNase P NTC reactions showed a Ct value of
approximately 45. What does this mean? ___________________________________
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Does this mean you cannot trust your data for the other reactions for this patient?
______ Why or why not? _________________________________________________
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What might have happened to result in this Ct value for this reaction? ___________
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6. In Patient #2, there was a positive reaction for both the InfA reactions and the
SW H1 reactions. How is this possible? Does this mean this patient is infected with
two strains of flu? Why or why not? ________________________________________
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7. What can you say about Patient #3’s Ct values? What would you report to this
patient as his/her diagnosis? _______________________________________________
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Section 5: Reporting Results
The CDC does not require reporting of any Influenza A viral strains, including H1N1.
However, you must write diagnoses for each patient, and include a prescription, if
applicable.
1. Diagnosis and prescription for Patient #1 _________________________________
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2. Diagnosis and prescription for Patient #2 _________________________________
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3. Diagnosis and prescription for Patient #3 _________________________________
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4. Because the CDC does not require reporting of these viral strains, what does that
tell you about the number of actual cases in the US of H1N1 compared to the
number of reported cases of the same disease? ________________________________
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