Sun. Oct 2nd, 2022
Image of a man gesturing behind a lectern.
enlarge Centers for Disease Control and Prevention director Robert Redfield speaks at a press conference about the 2019 nCoV outbreak.

As the newly discovered coronavirus has spread rapidly beyond its origin in China, health authorities around the world had to rapidly develop testing capabilities. In the United States, that task has been performed by the Centers for Disease Control (CDC), who have published their methodology and are currently applying for an emergency waiver to enable medical testing facilities to conduct these tests.

But if you’re not familiar with the tools of molecular biology, the CDC’s testing procedure might as well be written in another language. What follows is a description of how to go from an unknown virus to a diagnostic test in less than a month.

start from nothing

When the Chinese health authorities were first confronted with the outbreak, it had a disturbing notoriety. They had already dealt with a similar set of symptoms during the SARS outbreak in the early 2000s and had seen the spread of MERS a decade later. Thanks to these and related viruses, we had a detailed description of the structure of the typical coronavirus genome as early as 2005. That knowledge would undoubtedly be essential for the first step in the development of a rapid diagnostic test: characterization of the genome of the new virus, 2019-nCoV.

Knowing what the average coronavirus looks like has allowed us to identify areas that don’t change much during the evolution of new members of this family of viruses. And that allows us to obtain sequences of its genome without isolating the virus first.

The first challenge in sequencing a coronavirus genome is that it is made of RNA rather than DNA. Most of our tools for working with nucleic acids are specific to DNA. Fortunately, we’ve discovered an enzyme called “reverse transcriptase” that takes RNA and makes a DNA copy of it – transcription is the copying of DNA into RNA; this enzyme does the opposite, hence the name. (Reverse transcriptase was first identified in other RNA viruses that must be copied into DNA as part of infection.) Using reverse transcriptase, researchers were able to make DNA copies of parts of 2019-nCoV as a first step in studying its genome.

But reverse transcription of samples from infected individuals would simply create a jumble of DNA fragments of everything present: the patient’s own cells, harmless bacteria, and so on. Fortunately, DNA sequencing and analysis techniques have become so advanced that it’s now possible to sequence the whole mess, irrelevant stuff and all, and let computers figure out what’s there. Software can take what we know about the average coronavirus genome and identify all sequence fragments that look like they came from a coronavirus. Other software can determine how all these fragments overlap and then put them together, creating a nearly complete coronavirus genome.

At this point, the Chinese health authorities recognized that the virus involved in these infections was new, and they quickly published the genome sequence of the virus so that other health organizations could be prepared.

From genome to sampling

To create a diagnostic test specific to 2019-nCoV, researchers had to look for parts of the genome that don’t change rapidly during the evolution of the coronavirus, but that have changed enough in this branch of the family that they can become seen as its distinctive signature. Those sequences can be used to design a way to amplify a piece of the 2019-nCoV genome using a technique called the polymerase chain reaction, or PCR.

We won’t go into all the technicalities of how PCR works, in part because we’ve already done that. To understand the diagnostic test, all you need to know is that you need to design two small pieces of DNA that fit (meaning they can base pair with) two parts of the genome that are a few hundred base pairs apart. These small pieces of DNA are called ‘primers’. PCR will amplify the stretch of DNA between the two primers.

It does this by making the DNA go through heating and cooling cycles in the presence of enzymes that copy DNA. Each time through the cycle, the enzymes can make two new copies of the section between the primers. Using this process, it is possible to take a piece of DNA that is extremely rare and make billions of copies of it.

But PCR works with DNA, and the coronavirus is made of RNA. So we need to use reverse transcriptase first before trying to perform PCR. Fortunately, companies have developed solutions that contain all the enzymes and raw materials that both reactions require, enabling coupled reverse transcriptase-PCR reaction mixtures. The combination of reactions is called RT-PCR. With the right primers, RT-PCR can allow us to start with a chaotic mix or RNA and leave us with a large number of copies of a specific piece of the 2019-nCoV, provided they were present in the original sample. .

The problem is that PCR is so sensitive that it can amplify even small errors: primers sticking to a distantly related sequence, a distantly related coronavirus in the sample, or even contamination from the previous sample. While these errors are rare, the exponential amplification provided by PCR can eventually cause one to dominate the sample. Fortunately, people have come up with a way to take advantage of the rarity of these errors.

get real

If the correct sequence for the primers is present – meaning 2019-nCoV is present in the sample – the amplification will usually start with the very first cycle and grow rapidly. Errors, on the other hand, can take several cycles and the gain therefore lags a little. To find out when 2019-nCoV is really present, we need to identify when the amplification happens quickly and when it lags. We need to observe the progress of the PCR cycles in real time.

To this end, scientists developed a dye that only fluoresces when double-stranded DNA is present. When the reaction starts, there is very little of it, so the fluorescence is low. But as more amplifications take place, the glow rises rapidly until there is so much DNA that the difference between cycles becomes impossible. If the gain starts early, this rise and saturation occurs early; if it depends on an error, it will take longer to see them.

So real-time RT-PCR (RRT-PCR, for those keen on jargon) gives us a way to determine if a PCR amplification occurs because our sequence of interest is present. (It can also be used to get an estimate of the relative amount of that sequence present, but it’s not necessary for this test.)

Because this is such an important technique, companies have developed products around it. You can buy the fluorescent dye, enzymes, etc., as well as a machine that integrates the thermal hardware to run the reaction and has a light sensor to monitor the fluorescence. If you want to do this yourself, suitable hardware seems to be available on eBay for somewhere in the neighborhood of $2,000.

Kits aren’t all you need

However, if you look at the CDC’s instructions, you’ll see little discussion of the hardware or enzymes. Instead, you’ll find a discussion of ways to avoid contamination. If a facility tests many samples, there will be no shortage of 2019-nCoV DNA, both from the samples and from the previous PCR reactions. Given the ease with which PCR can amplify rare sequences, this can pose the risk of hordes of false positives. So the CDC reflects a set of best practices such as preparing RT-PCR reaction mixtures with a separate set of hardware than that used to process samples.

Another big part of the instructions goes into the details of the correct controls. Some of these omit important reactants, such as enzymes or sample RNA, to ensure contamination does not produce false results. This will tell you if you should trust positive results. There’s also a positive check, to make sure there’s nothing wrong with the reaction mix, so you know if you can trust negative results.

That said, the tests won’t be final. We don’t yet know enough about the virus life cycle to know the dynamics of infection: how long after infection does the virus become detectable and when does that compare to the onset of symptoms. It is very possible that asymptomatically infected people will not have enough virus for this test to pick up the virus consistently. So the CDC still advises caution with people believed to be at risk for infection.

But as the number of person-to-person transmission cases appears to be on the rise outside of China, testing without the need to send samples to the CDC headquarters in Atlanta could significantly aid our ability to respond to a rapidly evolving outbreak.

By akfire1

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