Diagnosing patients infected with SARS-CoV-2 during the COVID-19 (coronavirus disease) outbreak
Diagnosing patients infected with SARS-CoV-2 during the COVID-19 (coronavirus disease) outbreak
Part I
Patients with “flu-like” symptoms who test negative for influenza or other respiratory pathogens are key candidates for coronavirus testing. The more reliable COVID testing now used by public health labs around the country is based on PCR, an indispensable molecular biology technique used for the past few decades. While PCR is similar to DNA replication inside cells, there are some differences. It helps to review the key steps of DNA replication and note how PCR is the same or different.
1. First summarize the key steps of replication and the “key players” for each step. Your textbook has a good summary of the key players of DNA replication.
2. Watch the PCR video provided.
3. Make a list in the last column noting if the key players of PCR are the same or different from replication.
| Key Players of DNA replication | Summary of DNA Replication | Key Players of PCR |
The PCR procedure must be slightly modified for COVID diagnostics since the “template” being copied is not DNA. Identify the enzyme that can make DNA from an RNA template? Hint: It’s actually a viral enzyme!
The testing kits use the above enzyme to first produce a DNA template that is during the PCR process.
Part II
The idea behind PCR-based diagnostics is that a very small number of microbial genomes in a patient sample can be multiplied by PCR and more easily detected by the clinical team managing the patient’s care. Also, genetic-based diagnostics are very useful for viral infections because we don’t have biochemical tests, etc. to distinguish one virus from another (remember, viruses are metabolically inactive). However, a lot of work goes into the development of these tests. For instance, PCR requires primers that are complementary to the viral genome that is being copied.
1. If primers are complementary to the target genome, what must scientists know to design primers that bind to the viral genome to be copied? (I mean this to be a general question so please don’t look up the details of designing primers like primer length or “G/C” content.)
Part III
Coronaviridae is a virus “family.” There are actually many different coronaviruses within this group, including less pathogenic virus strains that cause the common cold. Many people in this course have likely been infected with one of these other coronaviruses.
A common feature of coronaviruses and other RNA viruses (think influenza virus, HIV, etc.) is that they mutate frequently. This is because the viral enzymes that copy the genome during replication can’t fix mistakes.
1. Given this information, how might we be able to distinguish the SARS-CoV-2 strain from some of the other, less concerning, coronavirus strains?
Part III
Luckily, the complete coronavirus genome sequence from the first known patient infected with SARS-CoV-2 was published on January 10th 2020, just a short time after the patient had been hospitalized in China with respiratory symptoms. They did this so quickly using nanopore sequencing. Watch a video about it; it’s pretty cool. Scientists compared the genome sequence from the new coronavirus (SARS-CoV-2) with the genome sequences from several other coronaviruses and they did identify a sequence unique to the SARS-CoV-2 strain. This unique sequence was in the N gene of the viral genome.
1. Look up what protein is made from the N gene of the virus.
If the unique N gene sequence can be copied with PCR and detected in a patient sample, this is proof that the virus is present in that patient. They’re infected!
Part IV
PCR primers are designed to only replicate the N gene sequence of the viral genome. Part of the N sequence we want to amplify is shown below. Typically, you design two primers, one to bind to each strand of the dsDNA. Copies are made from each strand so you get twice as much DNA from the PCR process!
Potential primer locations are noted by the nucleotide sequences shown below.
The PCR needs to make copies of the nucleotides shown in the middle (“87 nucleotides”).
Remember the direction that DNA polymerase synthesizes new DNA strands. Select the two locations for the primers to bind and then fill in the correct sequence below the DNA sequence shown. You should have selected one location on each strand.
5’-CACATTGGCACCCGCAATC———————-87 additional nucleotides—————–CAAGCCTCTTCTCGTTCCTC-3’
3’-GTGTAACCGTGGGCGTTAG———————87 additional nucleotides—————–GTTCGGAGAAGAGCAAGGAG-5’
Indicate the direction that the DNA polymerase (Taq polymerase) will move after binding to the primers.
Part V
After you design the PCR primers, you run the PCR on fluid from the patient’s nasal swab. Next, you need to evaluate the results. PCR makes billions of copies of just one sequence in a sample. Since you know the sequence, you also know the length (number of nucleotides) of the region you copied. I counted for you! There are about 130 nucleotides in the N gene fragment. This is typically stated as 130 base pairs, or 130 bp.
How can you visualize DNA and estimate its size? Load the DNA sample into an agarose gel (similar in consistency to jello) and apply an electric current to the gel. The DNA is charged and will move through the gel. The longer the DNA fragment, the more slowly it moves. The DNA is visualized by adding a fluorescent dye to your sample that sticks to DNA. When you look at the gel under UV light, the DNA should glow.
1. What is the charge of a DNA molecule?
2. Based on #1, would you expect DNA to be drawn to the (+) or (-) electrode of the gel electrophoresis chamber?
Evaluate the gel provided below. Use the notes to help you read the gel:
· DNA is loaded in the wells (little pockets) at the top. The wells are the dark spaces under the numbers.
· DNA moves from the wells into the gel. The smaller the DNA the more quickly it moves.
· The samples on the ends are DNA ladders. We know the sizes of each of those bands and can use them to estimate the size of the DNA in the patient samples.
1: DNA ladder
2: Positive control
3: Patient 1 sample
4: Patient 2 sample
5: Patient 3 sample
6: Negative Control
7: DNA ladder
300 bp
100 bp
1. Why are positive and negative controls needed for PCR (or any test)?
2. Initially, test kits sent out by the CDC were flawed. The negative control sometimes showed up as positive. What misdiagnosis would potentially occur with this mistake? (Over or underdiagnosis?)
3. The CDC ultimately said to use the test anyway. Why might this be the case, given the current situation?
4. How would you evaluate the results above? Do any of the patients have SARS-CoV-2?
Part VI
The results of PCR-based procedures can be reported in multiple ways. Like the ELISA, PCR can be quantitative. The intensity of the fluorescence corresponds to the number of DNA molecules in the sample. This is helpful. We can determine the presence of the viral genome in a patient sample and we can also determine how many genomes are there. Why does this matter? Well, it is one tool that helps evaluate the effectiveness of a treatment or the patient’s own immune response to the microbe. For example, if the “viral load” (number of viral genomes) decreases following treatment but stays the same in untreated individuals this is one piece of data the treatment is effective. Some quantitative PCR data is shown below.
1. This is PCR data from three patients that were tested for COVID. Each colored line (blue, red, or green) represents the PCR results from a different patient. The vertical black line is considered the “threshold” for detection. Since PCR occurs through multiple cycles, if there are no results by the threshold cycle the data is considered negative. Evaluate the data (and especially the axis labels). Can you identify if any patients are positive or negative for COVID? How did you determine this? (Assume the controls worked properly even though they aren’t shown.)
