Marcus stepped out of his car and into the sunshine, ready to head into the hospital for another day of work in
the molecular diagnostics laboratory. Having graduated six months ago from college, he felt very fortunate to have
found a great job that would give him valuable experience until he was ready to apply to medical school. Marcus had
majored in biology and spent his senior year working in a research lab studying the genes that control early embryonic
development in zebrafish. In his current job, he was studying genes again, but this time for a completely different
purpose: to find genetic changes in patient DNA that could be contributing to their disease. Entering the laboratory
wing of the hospital, Marcus thought, Working in the molecular diagnostics lab sure is different from when I was doing
experiments in the research lab!
2
What made Marcus think that? What exactly is a molecular diagnostics lab, and how does it differ from a research lab?
A molecular diagnostics laboratory is just one of several types of laboratories that exist in hospitals. These laboratories
are also referred to as medical laboratories, or clinical laboratories. The term “clinical” refers to the actual diagnosis and treatment of patients.
The following resources may help you to answer the questions below.
• Food and Drug Administration (FDA). Tests used in clinical care.
<https://www.fda.gov/medical-devices/vitro-diagnostics/tests-used-clinical-care>
• American Association for Clinical Chemistry (AACC). Where lab tests are performed.
<https://labtestsonline.org/articles/where-lab-tests-are-performed>
• American Association for Clinical Chemistry (AACC). Collecting samples for laboratory testing.
<https://labtestsonline.org/articles/collecting-samples-laboratory-testing>

Questions
1. What types of samples might be collected from patients to use for laboratory testing?
2. What are some examples of situations where a doctor might order a lab test to aid in the diagnosis and treatment of a patient?

The Power of a Test:
How COVID-19 Is Diagnosed and Who Does It
by Alison Kieffer, Emaly J. Piecuch, Christina Vallianatos, & Sarah A. Wojiski Genomic Education
The Jackson Laboratory, Bar Harbor, ME

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There are many rules and regulations in place in order for a clinical laboratory to conduct testing. All clinical labora–
tories are regulated by the government, through the Clinical Laboratory Improvement Amendments, or CLIA. All
clinical laboratories must be CLIA-certified. To obtain certification, a laboratory needs to provide evidence that the
tests that they perform meet quality control standards, that the personnel performing the tests are adequately trained,
and that the equipment being used for the test is functioning correctly and is properly calibrated.
The following resources may help you to answer the questions below.
• American Academy of Family Physicians (AAFP). Clinical Laboratory Improvement Amendments (CLIA).
<https://www.aafp.org/practice-management/regulatory/clia.html>
• Food and Drug Administration (FDA). Clinical Laboratory Improvement Amendments (CLIA).
<https://www.fda.gov/medical-devices/ivd-regulatory-assistance/clinical-laboratory-improvement-amendments-clia>
• Centers for Medicare and Medicaid Services (CMS). How to obtain a CLIA certificate.
<https://www.cms.gov/Regulations-and-Guidance/Legislation/CLIA/Downloads/HowObtainCLIACertificate.pdf>

Questions
3. Why does the government regulate clinical laboratories? Why is CLIA certification required?
4. How does the work of a clinical laboratory differ from the experiments being conducted in a research laboratory?
Why don’t research laboratories have to comply with CLIA regulations?

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Part II – Diagnostic Testing
For weeks, Marcus had been checking the Centers for Disease Control and Prevention (CDC) website in the evenings
after work. The virus, referred to as SARS-CoV-2, was spreading across the globe and seemed to be taking hold in the
United States, with cases of the associated respiratory disease COVID-19 increasing daily. The World Health Organi–
zation (WHO) had officially classified the outbreak as a pandemic, and large numbers of cases were being reported in
the state where Marcus lived.
When he arrived for work this morning, the laboratory director, Dr. Elaine Cordozo, called an emergency staff meet–
ing. When everyone had gathered in the conference room, she made an announcement. “Working with our state’s
health department, we have been given the important job of serving as a sample processing and testing laboratory for
COVID-19 infection. We will begin preparations for conducting this testing immediately and will commence testing
of patient samples as soon as possible.”
The next several days were a blur for Marcus and his colleagues. They worked quickly to get the laboratory testing set
up. Finally, the day arrived that they would commence with COVID-19 diagnostic testing.
2
How does COVID-19 testing work? How do healthcare professionals make a diagnosis of COVID-19 infection? Let’s
follow Marcus through the process.
Read the following graphic article to learn about the SARS-CoV-2 virus and how it infects the human body:
• Corum, J. and C. Zimmer. 2020. How coronavirus hijacks your cells. The New York Times.
<https://www.nytimes.com/interactive/2020/03/11/science/how-coronavirus-hijacks-your-cells.html>
Watch this video to learn about the COVID-19 testing process:
• The Jackson Laboratory. 2020. COVID-19 testing process. Running time: 1:15 min.
<https://youtu.be/ORRLyCZpIus>
Carefully read the CDC test protocol outlined in Table 1 below to learn about how SARS-CoV-2 is detected after the
sample is collected from the patient and received by the clinical lab:
Table 1. Centers for Disease Control (CDC) SARS-CoV-2 coronavirus diagnostic test protocol for CLIA laboratory.
Purpose How it works Notes
Stage 1 Nucleic acid
extraction
To isolate viral
RNA from a
patient sample
All nucleic acids are first extracted from
the sample. Next, viral RNA is specifically
isolated from other sources of RNA.
Viral RNA is isolated based on
size. Viral RNA genomes are much
larger than RNA from other sources:
the SARS-CoV-2 RNA genome is
~30,000 bases long; human RNA is
~100s bases long.
Stage 2a Reverse
transcription
To convert
viral RNA into
cDNA
Viral RNA isolated in Stage 1 is read by a reverse transcriptase enzyme, which works to make complementary DNA (cDNA) copies of the RNA template.
The viral RNA from the patient sample must be amplified by PCR (Stage 2b) in order to be detected. PCR requires a DNA template, therefore, the RNA virus first needs to be converted to DNA.

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Stage 2b Quantitative
PCR To amplify, detect and quantify the virus-specific sequence in a sample PCR amplification using cDNA gener–ated in Stage 2a as template. Three fluorescent primer probe sets are used to detect distinct regions of the viral genome of SARS-CoV-2. A special PCR machine with a fluorometer detects fluorescence generated when primers find their target sequence and target DNA is amplified.
Fluorescence is quantified at every cycle of the PCR process in order to determine the amount of virus present. As PCR is used to amplify the target
sequences, more fluorescent probes can bind to the newly generated PCR
products, and more fluorescence is emitted and detected.
Stage 3 Data analysis To generate
diagnostic report
Analyze graphs of quantitative PCR results (fluorescence data and PCR cycle number) for each sample to determine if
it is SARS-CoV-2 virus positive, negative, or ambiguous.
The fewer cycles it takes to detect, the more abundant the viral genome is in the sample. Cycle number and level of fluorescence all contribute to
interpreting results as strong positives, strong negatives, or ambiguous.

Questions
1. After reviewing the protocol above, what terms have you heard of before?
2. After reviewing the protocol above, what terms are
unfamiliar to you?
Read the following graphic article to learn about the SARS-
CoV-2 genome:
• Corum, J. and C. Zimmer. 2020. Bad news wrapped in
protein: inside the coronavirus genome. The New York Times.
<https://www.nytimes.com/interactive/2020/04/03/science/
coronavirus-genome-bad-news-wrapped-in-protein.html>
Let’s take a closer look at the equipment used and at each stage of the protocol to better understand how a sample is processed and analyzed for COVID-19.
General Equipment Used for Sample Preparation The essential molecular biology equipment used in a diag– nostic lab is depicted in Figure 1. After individual patient samples arrive at the diagnostic lab, they are processed in small tubes stored on tube racks. Micropipettors are used to draw up liquid solutions used in the various steps of this protocol. Multiple samples can be handled in parallel together on microplates to save time. These plates can hold 96 different samples, and are loaded onto a special PCR machine used for the amplification and detection of the target sequence.
Figure 1. Materials Used in the Diagnostic Lab.

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Figure 2. Steps in Processing a Patient Sample. The steps required to isolate viral RNA for diagnostic testing from a patient sample are shown. In step one, the different biological entities obtained from a swab are depicted. In step two, all nucleic acids
are released into solution. Step three shows viral RNA, isolated and ready to analyze.Note the images are not to scale.

Stage 1 – Nucleic Acid Extraction
After the sample arrives in the diagnostic lab, the first step is to specifically isolate the genetic material of the virus from
all of the content found in a patient’s sample. In this instance, SARS-CoV-2 uses RNA as its genetic material, so viral
RNA must be extracted and isolated. The process is as follows and shown in Figure 2:
1. Obtain the patient sample.
2. Expose all of the genetic material found in the sample.
3. Purify the viral RNA and discard all other contents.

Questions
3. What samples are typically collected from patients suspected of coronavirus exposure?
4. Name three sources of nucleic acids you expect to find in a patient sample.

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Figure 3. Steps in Reverse Transcription (RT). The molecular biology steps in the process of reverse transcription (RT) leading to the generation of complementary DNA (cDNA) from an RNA sample are shown. This process allows for the amplification of the sample in Stage 2b, quantitative PCR (qPCR).
Stage 2a – Reverse Transcription (RT)
Once the RNA from the virus is isolated, it is ready to be amplified, detected, and analyzed. The first half of Stage 2 (Stage 2a) requires the viral RNA to be
converted to complementary DNA copies, called cDNA for short. This process of making a DNA copy of RNA
is called reverse transcription. This is accomplished by a special enzyme called reverse transcriptase, which is not
normally found in living cells. Reverse transcriptase was actually discovered in, and isolated from, RNA viruses.
The process is as follows and is shown in Figure 3:
1. The viral RNA isolated from Stage 1 is in a solution.
2. The solution contains short single-stranded DNA oligonucleotides, called primers, that bind to the  RNA sequence.
3. The solution also contains free DNA nucleotides (A, C, T, and G).
4. The solution also contains the reverse transcriptase (RT) enzyme. The RT enzyme uses the primers to begin adding nucleotides complementary to the RNA.
5. The RT enzyme works along the RNA template, reading the RNA sequence and adding the complementary DNA nucleotides.
6. The RT enzyme makes an exact DNA copy of the  RNA template. It does this for all RNA present.
7. The cDNA is now ready for amplification by PCR.

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Figure 4. The Central Dogma of Molecular Biology. Use your knowledge of the central dogma to match each term to the numbers in the figure and identify how reverse transcription (RT) fits into this process.
___ cDNA
___ DNA
___ Protein
___ RNA
___ Reverse Transcription
___ Transcription
___ Translation
Figure 5. Steps in Quantitative PCR (qPCR). The steps in the
process of the quantitative PCR (qPCR) assay are shown.
This process leads to the amplification and quantification of a cDNA sample.
Stage 2b – Quantitative PCR
Watch the following video about quantitative PCR (qPCR):
• Overview of qPCR. Produced by New England
Biolabs, 2016. Running time: 2:44 min.
<https://youtu.be/1kvy17ugI4w>
The second part of Stage 2 serves to amplify and detect the cDNA generated in Stage 2a. Like traditional PCR,
quantitative PCR (qPCR) cycles through temperatures in order to amplify few DNA copies into many DNA copies.
Unlike traditional PCR, qPCR is able to determine ex–actly how many copies of DNA are present in the sample.
The process is as follows and is shown in Figure 5:
1. The cDNA generated in Stage 2a is used as a template for the qPCR amplification. Primers find the target sequence to help the polymerase start to amplify
and make more copies. Fluorescent probes are in the solution that will detect the target sequence as more copies are made during each cycle of the PCR.
2. As in a traditional PCR, the target sequence is amplified exponentially.
3. As more copies of the target sequence are made, there is more chance for the fluorescent probes to bind. This fluorescence is detected by the fluorometer
inside the qPCR machine. Since the cDNA is a DNA copy of the RNA from the virus, the more viral
sequence in your sample, the more fluorescent probes will bind. With every cycle you will detect morefluorescence.
Activity 1
Recall the central dogma of molecular biology: When DNA is made into RNA it is called transcription. When RNA is made into protein it is called translation. Where does reverse transcription fit in this process? Match each keyword to the number in Figure 4 below.

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Questions
5. Compare and contrast traditional PCR with quantitative PCR.
6. How can we ensure we are only amplifying and detecting viral sequences, and no other sequences that may be in the tube?
Figure 6. Quantitative PCR (qPCR) Report. Depicted here is an example of a qPCR assay report. This is the data that needs to be interpreted for diagnosis. A positive control sample is used to determine the fluorescence intensity of a known sample. A negative control sample is used to determine the fluorescence intensity of a sample that is known to have no viral RNA. The fluorescence of a patient sample can be compared to that of the control samples to determine if the sample is positive or negative. Sometimes, sample fluorescence can be intermediate, which is interpreted as an ambiguous patient sample (unsure if the sample is positive or negative).
Stage 3 – Data Interpretation
The graph below in Figure 6 shows the type of data collected from a quantitative PCR (qPCR) assay. Let’s break down
the components of the graph to analyze the results.
• Fluorescence (y axis) is tracked and reported for each sample across all cycles of the PCR assay (x axis).
• The threshold of detection (Line D) is the amount of fluorescence needed to distinguish true fluorescence signal from background signal from the assay.
• A positive control (Sample A) is included as a reference for what a virus-containing sample should look like;
positive samples should create a high fluorescence signal, far above the threshold of detection.
• A negative control (Sample E) is included as a reference for what a sample without virus should look like; negative
samples should yield a low fluorescence signal, below the threshold of detection.
• In this graph, three patient samples (Sample B, Sample C, and Sample F) illustrate different outcomes from this assay.

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Figure 7. Laboratory Testing Process for Two Different Patient Samples. Depicted here is an
example of diagnostic sample processing for two different patient samples. In Stage 1, notice
the amount of RNA in each tube is different. In Stage 2, notice the amount of cDNA that is
synthesized during the RT step is different. In Stage 3, notice the amount of amplification
during the qPCR varies. Due to these factors and other variables in sample processing, you can
see that the diagnostic result for each sample is quite different.
To summarize, let’s look at two patient samples and follow the laboratory process, as depicted in Figure 7:
1. Two samples are received in the lab. Viral RNA is isolated from each.
2. The RNA is converted to cDNA via reverse transcription. The cDNA is amplified via quantitative PCR and the
fluorescence from the primers is detected.
3. Data is graphed from the qPCR assay. Sample 1 shows a high signal detected early in the cycle run, similar to the
positive control. Sample 1 is reported as SARS-CoV-2 positive. Sample 2 shows a low signal below the threshold
of detection, similar to the negative control. Sample 2 is reported as SARS-CoV-2 negative.

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Figure 8. qPCR Reports for Three Different Patient Samples. Use your knowledge of the diagnostic testing process to make a diagnosis in the three patients.
Activity 2
Figure 8 shows three examples of quantitative PCR results from three different patients. Use what you have learned about the negative control,
positive control, and threshold of detection to la–bel the lines. Notice the difference in the number
of cycles (x axis) that each sample takes to peak in its fluorescence (y axis). Using your knowledge,
interpret results of each test by indicating “posi–tive,” “negative,” or “ambiguous” on the lines
provided below.
Patient #1 Result: _________________
Patient #2 Result: _________________
Patient #3 Result: _________________

Question
7. What could contribute to ambiguous results from samples? Hint: Think about differences in the viral load (the total amount of virus inside a person) between people, differences in sample collection methods, the differences in sample preparation methods. Can you suggest two factors that may contribute to an ambiguous test result?