The basic test for virus is called RT-qPCR, i.e. first RT (reverse transcription) and then a modification of PCR called quantitative PCR. We'll talk about PCR first. Then I'll go back for RT and finally for the q.
I suppose most people know something about PCR. PCR is short for polymerase chain reaction, we'll see the reason for that name at the end.
level 1: basics
You start with DNA, which is usually though not always double-stranded. If that statement is confusing, you can read up on DNA in wikipedia (and don't forget to go here to see a famous pic). Hence, we talk about the fundamental unit of DNA as a base pair.
DNA has polarity, the two ends of each strand are chemically different. DNA synthesis or replication proceeds with the polarity 5' to 3'.
In a diagram, the 3' end is conveniently distinguished with an arrowhead. That's the direction of chain growth during synthesis.
In a nutshell, in PCR you may start with any old double-stranded template DNA. It could be a known DNA like a plasmid or it could be from a clinical sample or a crime-scene specimen, or something else, as we will see.
In addition, you will need some sequence information about the template to design two short primers that have the same sequence as chosen target regions in the template. The positions where the primer sequences are found in the template are indicated by the dashed lines in the figure above.
The primers are present in vast excess to the template, though still in single-digit nanogram amounts.
You put the template and primers in a tube with some other stuff, stick that tube in a "PCR machine" and press a button. 2-3 hours later, at the end of the PCR process, you have product, often lots of it. The reaction product(s) can be analyzed on a gel, like this one.
level 2:
Template DNA might be anywhere from 100-200 bp (base pairs) in length up to as large as a chromosome. As long as there are at least a hundred or so copies of template in the reaction tube, it's going to work. It is OK if the template is just one strand, but it has to be DNA.
Primers are synthesized chemically. When I was a student, there were specialists (chemistry graduates) who did this by hand, one base at a time. It was a dark art. Now they come in the mail.
The reaction requires more than just primer and template. You need a special enzyme (DNA polymerase), plus the four nucleotides that assemble into the new DNA (technically, deoxyribonucleoside triphosphates). Also, you need the right buffer to establish pH, set Mg2+ at the right concentration, and EDTA to chelate bad stuff.
Here's a diagram of a base (technically, a nucleotide) being added to a growing primer.
That HO group in red is a 3' hydroxyl, and it's the position where the incoming base is attached.
The key to the process is the idea of cycling the temperature. That's Kary Mullis's innovation.
You first heat to near boiling (95°C), to denature the template (make the two strands come apart). Then cool to the annealing temperature, somewhere between 55-60°C. During this stage the primers bind to their locations on the template. Then increase to 72°C and let the synthesis proceed. (The reactions proceed under mineral oil, otherwise all the water would turn into vapor and condense on the lid of the tube).
You might think 60 C is hot, and it is. (9/5 * 60) + 32 = 140°F, that's about the temperature of over-done roast beef or under-done chicken. 72°C is, of course, hotter.
For a DNA polymerase to function at that temperature (and not come apart at 95°C), it has to be special. Hence Taq polymerase, from Thermus aquaticus (or various others that have been used).
So the typical procedure would be to do 30 or 40 cycles of the the 3 temperatures and then analyze what you've got.
This all happens in a machined aluminum block that accepts 0.5 mL tubes and is hooked up to heating/cooling and can change temperature quite rapidly. The device is programmable so that running a profile is a matter of pushing two or three buttons. They are called thermal cyclers.
level 3:
There are a couple different reactions going on, which happen with different kinetics of product accumulation.
In cycle 1, this is all that happens. But it happens in every cycle, so this extended product accumulates linearly. These products can be reasonably long, and end at random positions when the temperature is raised back up to 95°C at the end of the cycle, or because the template is randomly broken (sheared).
(Note: an identical process begins with the red strand of the template, but is not shown).
In cycle 2 and beyond, the same reaction from above occurs, but another thing happens, the extended products themselves serve as templates.
The third reaction dominates.
Here, product synthesized in a previous cycle serves as template for synthesis of another copy. Because products serve as templates, the kinetics for this are exponential.
Assuming for simplicity that the efficiency is 100%, the amount of product doubles every cycle. So roughly speaking, in 30 cycles you could get as much as 2^30 times as much product as template that you started with. That's 2 to the power 30, about one billion. Hence, the reaction is called a "chain" reaction.
This usually doesn't happen for various reasons, but you can make quite a bit of product, hundreds of nanogram.
q and RT
So briefly, the other two points. The genome of SARS-CoV-2 is RNA. DNA polymerases will not copy an RNA template. However, a very well-known virus that has an RNA genome replicates to a DNA intermediate. That virus is HIV and the family is called Retroviridae, because genetic information is flowing backward, from RNA into DNA.
Retroviruses have an enzyme packaged inside the vision called reverse transcriptase which copies the RNA genome into DNA. Hence, the first step for RT-qPCR is a reverse transcription reaction.
The last thing is the q, for quantitative. The original design was to use gel electrophoresis to analyze the product, like the gel shown above. But, this is time-consuming and it's not quantitative, for technical reasons. For diagnostics, real-time or quantitative PCR is used.
So modern labs have PCR machines with optics. A beam of light travels through each tube as the reaction proceeds. The reaction mixture is modified so that there is a fluorescent signal from the product, which is quantified by a detector. It's very cool technology: there is a fluorophore and a quenching agent attached to a probe, when the probe binds the PCR product, the quencher is separated from the fluorophore and it lights up.
The usual description for a PCR reaction used in a clinical test is to talk about the Ct, the threshold cycle. That is the cycle during which product is first detected. (It can be determined accurately by extrapolating from data obtained in later cycles).
The smaller Ct is, the more template was present at the beginning. A change in Ct of -1 unit means a doubling of the the amount of virus present at the beginning.
Finally, we should remember that detection of viral genome RNA does not make strong predictions about how much viable virus is present, for several reasons. Infections with most eukaryotic viruses produce large numbers of viral particles that are not able to go on to cause another infection. Without researching, I don't know the number for coronaviruses, but it can be as small as 1 viable virus/1000 virus particles for poliovirus (link). Also, if viral RNA is intracellular and not packaged into a vision, it won't be infectious.
That's why when you see studies showing people testing positive for SARS-CoV-2 viral RNA at 14-21 days it isn't anything to be concerned about. Most authorities say that people are not infectious beyond about 5 days after they become symptomatic.
I like Carl Zimmer's writing very much, but in this article he says that "[the PCR] recipe involves many steps, which are typically carried out by trained technicians." That's just silly. If you have the master mix and the RT product, you mix them together, put them in the cycler and push a button. I could teach my granddaughter Ella to do it in less time than it took you to read this.
