Coronavirus testing

Objectives:

The most widely used diagnostic technique for COVID-19 uses a variation of PCR.  It's quite different from the PCR approach you use in the Bio 6B lab, but it builds on the same principles.

By the time you complete this unit, you should be able to:

• Explain how SARS-CoV-2 RNA testing works.
• Compare and contrast standard PCR, RT-PCR, and RT-qPCR.

Covid tests are based on PCR

Having completed a unit on the polymerase chain reaction (PCR), you know how that process works and why it’s useful. PCR allows lab workers to specifically copy a desired DNA target, even when it’s present in tiny quantities and mixed with other DNA. Thus, PCR is an ideal technique to use for detecting viruses such as SARS-CoV-2, the virus that causes Covid-19. However, there are some problems that need to be solved to make an effective PCR test:

RNA

The coronavirus genome is RNA; PCR is a DNA technique. A DNA copy of the RNA must be made before PCR begins.

Product analysis

How will you be sure that your PCR product is the correct one? In the Bio 6B lab, you would normally perform PCR and then run your PCR products on an electrophoresis gel to determine their size (this is called end point PCR, because you run the reaction until it’s done and then analyze the result). If you see a PCR product of the expected size, it’s probably the correct product. However, it’s possible to get the wrong PCR product, which just happens to be the same size as the intended product.

Speed

For a virus diagnostic test, running each sample on a gel after PCR would take too much time, and it would introduce the possibility of mixing up samples. 

Quantitation

The traditional end-point PCR approach of gel analyzing products after PCR tends to give yes-or-no answers; you see the product or you don’t. For diagnostic tests, it’s better to have a quantitative measurement of the quantity of DNA product, reflecting the quantity of initial viral template RNA. This is called quantitation.

The PCR tests used for coronavirus solve these problems.

RT-PCR starts with RNA

RNA-based PCR tests start by making a DNA copy of the RNA. This process is called reverse transcription, because it’s the reverse of normal transcription (which makes RNA copies of DNA). Reverse transcription makes a single-stranded DNA copy (called a cDNA, or complementary DNA) of the RNA target. Reverse transcription requires a specific enzyme, called reverse transcriptase. The reverse transcription enzymes used in this process were originally cloned from retroviruses, which require this step for their life cycles.

The reverse transcriptase step requires specific DNA primers. Several different kinds of primers can be used:

• Specific primer: For example, a primer that binds to a segment of the SARS-CoV-2 genome. Only one primer is required with this approach, because the viral RNA template is single-stranded. This primer could be one of the two primers used in PCR, or it could be different.
• Random primers: If the goal is to copy all the RNA present in a sample, a selection of randomly selected primers is used. This approach is often used to make cDNA that will later be sequenced.
• Oligo(dT): Primers with a sequence consisting only of T. These primers will pair with the poly-AAAA nucleotide sequences commonly found on the 3' ends of mRNAs (including coronavirus mRNA).

Once the viral RNA template has been reverse transcribed, the resulting single-stranded cDNA product can then be amplified by standard PCR, using a DNA polymerase. This whole process is called RT-PCR, for reverse transcription PCR.

qPCR allows detection during PCR

For diagnostic tests, there’s a much better approach than the traditional end-point PCR method of running the PCR product on a gel for analysis after the PCR run is over. A technique called real-time PCR or quantitative PCR (qPCR) allows lab personnel to visualize the results of PCR as they happen. There is more than one technique for this, but they are all based on methods that produce fluorescence in the reaction tube as PCR products are synthesized. This approach uses a PCR machine (thermal cycler) that has a fluorometer built in, to detect the fluorescence as it is generated.

In one common approach, a probe is used along with the primers. The probe is an oligonucleotide (a short, single-stranded DNA molecule, similar to a primer) with a fluorophore attached. The fluorophore is a fluorescent molecule, meaning that it absorbs light at short wavelengths (such a UV or blue) and emits light at longer wavelengths (such as red). The fluorophore is on one end of the probe, but its fluorescence is undetectable, because the other end of the probe contains a quencher molecule that absorbs the fluorescence energy.

Once PCR begins, the oligonucleotide probe can specifically anneal (or hybridize) to the single-stranded target DNA. At this point, no fluorescence is visible, due to the quenching. The DNA polymerase begins DNA replication, starting at the 3' end of the primer. As the DNA polymerase copies the template, eventually it runs into the probe. The polymerase breaks up the probe DNA (in other words, the polymerase has exonuclease activity). Once the probe DNA is chopped up, the fluorophore is separated from the quencher, so the fluorophore can begin to emit fluorescence. The amount of fluorescence in the PCR tube will be proportional to the amount of degraded probe, which is proportional to the amount of PCR product produced.

Because PCR amplifies target DNA exponentially, the fluorescence level increases exponentially, producing a clear difference between a positive test (containing coronavirus template) and a negative test (no coronavirus, no PCR product, no fluorescence). The qPCR thermal cycler measures the fluorescence, and the relationship between the amount of starting virus RNA and the level of fluorescence can be determined experimentally.

The qPCR approach solves more than one problem. It allows detection of PCR products while the PCR run is happening, without requiring the product to be taken out of the PCR tube. This is much faster and more reliable, and it eliminates the chance that someone will mix up the samples when loading the gel. The fluorescence detection is very sensitive, allowing detection of tiny amounts of PCR product. Since the starting sample might contain very few RNA molecules, this is important. Finally, since the probe is a specific oligonucleotide that becomes fluorescent only when it binds to the correct PCR product, the probability of false positives (detecting the wrong PCR product) is greatly reduced.

For an excellent description of RT-qPCR, see A beginner’s guide to RT-PCR, qPCR and RT-qPCR. 

PCR requires controls

If the RT-qPCR test is reliable, a negative result means that the template RNA was not present, and a positive result means that it was present. Two kinds of controls are needed for PCR reliability:

• Positive control: A reliable template is added to the reaction, ensuring that a product is produced. If the positive control fails, something is wrong, and negative results in other reaction tubes can’t be trusted. In regular PCR, the positive control template is DNA, but for RT-qPCR, an RNA positive control template is needed so it can be a control for both the reverse transcription step and the PCR step.
• Negative control: No template RNA or DNA is added to the reaction, so no product should be produced. If a product is produced, it means that the reagents are contaminated with DNA or RNA, so the results of other reactions can’t be trusted.

As with any experimental procedure, the results can’t be trusted unless proper controls are used.

Multiplex PCR

Traditional PCR uses one pair of primers to amplify one target sequence. Multiplex PCR uses multiple primer pairs in the same reaction tube, to amplify multiple target sequences. This can be very useful for diagnostics. The two tests accepted by the CDC in the U.S. are both multiplex:

CDC Multiplex SARS-CoV-2 only test:

Amplifies several target sequences within the SARS-CoV-2 genome. This increases certainty that the correct target sequence is being identified, and makes the test more less likely to fail with mutant strains of the virus. In addition, one of the targets is a human RNA, which should always be present in nasal swab samples. If this positive control fails, it’s likely that the sample was not collected properly.

CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay: 

This assay simultaneously tests for both coronavirus and influenza A and B viruses. If a patient comes in with an apparent respiratory virus infection, the health care provider can use this test to quickly determine which viruses are present.

Thanks to the use of tags that emit fluorescence at different wavelengths, PCR machines can detect each product in a multiplex PCR separately.

Other countries use different tests, but they are also multiplex RT-qPCR.

Review

Terms & concepts

Terminology note: I have used the abbreviation RT-qPCR to mean reverse transcription quantitative PCR. I believe this is the most correct abbreviation, and is used by some of the companies that make supplies for this technique. Unfortunately, these terms are used inconsistently. For example, Campbell (Chapter 20) calls it qRT-PCR, and some sources use RT-PCR to mean real-time (quantitative) PCR. qPCR  is both real-time and quantitative.

• cDNA
• Controls: positive and negative
• DNA polymerase
• Fluorescence: fluorophores and quenching 
• Multiplex PCR: Amplifies more than one target sequence in one PCR reaction.
• Oligonucleotide: A short segment of single-stranded DNA or RNA. In PCR, the oligonucleotides are always DNA. The primers for all types of PCR are oligonucleotides. The probes used in qPCR are also oligonucleotides, modified with the addition of fluorophores and quenchers.
• Probe
• qPCR (also called real-time PCR; confusingly, sometimes abbreviated RT-PCR)
• Reverse transcriptase enzyme
• RT-PCR: reverse transcription PCR
• RT-qPCR 

Review questions

1. In the introduction, I mentioned several challenges posed by using PCR in a viral diagnostic test: working with RNA, PCR product analysis, speed, and quantitation. Explain how RT-qPCR solves each of these problems.
2. Explain the roles of positive and negative controls in PCR.

References & further reading

Videos

Overview of qPCR. New England Biolabs. Overview from a biotech supplier.

Probe-based qPCR. Sigma-Aldrich. It’s an ad with annoying music, but it has a good graphic depiction of the fluorescent probe mechanism in qPCR.

How TaqMan Works -- Ask TaqMan. Thermo  Fisher. This guy loves his TaqMan! It’s the most widely used fluorescent probe technology for qPCR, and this video shows how it works.

PCR, RT-PCR & RT-qPCR

A beginner’s guide to RT-PCR, qPCR and RT-qPCR. Adams, 2020. The Biochemist. Outstanding explanations and graphics. The best single article to read.

Basic Principles of RT-qPCR. ThermoFisher.

Real-time polymerase chain reaction. Wikipedia

The Science Behind the Test for the COVID-19 Virus. Mayo Clinic. This article offers a clear explanation of a test based on RT-qPCR.

Covid testing

Diagnosis in the Covid Reference. This article gives a comprehensive and up-to-date rundown of the various approaches and challenges in COVID-19 testing. The overall site is like a textbook for COVID-19.

Laboratory protocols (technical)

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Corman et al., 2020. Eurosurveillance. This technical article describes the standard testing procedure used in Europe. 

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR. Kudo et al., 2020. PLOS Biology. “The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction.” This short technical article describes a current test.