What is Reverse Transcription Polymerase Chain Reaction?
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
Blah Blooh Bleeh Blah. Right?
Well, this is the test that will tell us if a person has got the corona virus or not. So listen up!
The corona virus is in the form of RNA:
Coronaviruses, so named because they look like halos (known as coronas) when viewed under the electron microscope, are a large family of RNA viruses. The typical generic coronavirus genome is a single strand of RNA, 32 kilobases long, and is the largest known RNA virus genome. Coronaviruses have the highest known frequency of recombination of any positive-strand RNA virus, promiscuously combining genetic information from different sources when a host is infected with multiple coronaviruses. In other words, these viruses mutate and change at a high rate, which can create havoc for both diagnostic detection as well as therapy (and vaccine) regimens.
But as best as I can tell, detecting the corona virus becomes pretty difficult unless it turns into DNA, which can be done by a process called Reverse Transcription.
With the newly formed DNA, replicate it – have it reproduce a lot, basically. That’s where PCR comes in. And with that (and a fluroscent dye that is added to make detection easier) you have a sample that you can check for the presence of the corona virus.
The first, PCR, or polymerase chain reaction, is a DNA amplification technique that is routinely used in the lab to turn tiny amounts of DNA into large enough quantities that they can be analyzed. Invented in the 1980s by Kary Mullis, the Nobel Prize-winning technique uses cycles of heating and cooling to make millions of copies of a very small amount of DNA. When combined with a fluorescent dye that glows in the presence of DNA, PCR can actually tell scientists how much DNA there is. That’s useful for detecting when a pathogen is present, either circulating in a host’s body or left behind on surfaces.
But if scientists want to detect a virus like SARS-CoV-2, they first have to turn its genome, which is made of single-stranded RNA, into DNA. They do that with a handy enzyme called reverse-transcriptase. Combine the two techniques and you’ve got RT-PCR.
So, here’s how it works, best as I can tell:
That article I linked to from Wired has a more detailed explanation, including more detailed answers about the “how”, if you are interested. Please do read it fully!
Now, which kit to use to extract RNA from a snot sample, which dye to use, which PCR machine to use – all of these and more are variables. Think of it like a recipe – different steps, different ingredients, different cooking methods. Except, because this is so much more important than a recipe, the FDA wags a finger and establishes protocol.
That protocol doesn’t just tell you the steps, but it also tells you whether you are authorized to run the test at all or not. And that was, uh, problematic.
For consistency’s sake, the FDA opted to limit its initial emergency approval to just the CDC test, to ensure accurate surveillance across state, county, and city health departments. “The testing strategy the government picked was very limited. Even if the tests had worked, they wouldn’t have had that much capacity for a while,” says Joshua Sharfstein, a health policy researcher at Johns Hopkins School of Public Health and the coauthor of a recent journal article on how this testing system has gone awry. “They basically were saying, we’re going to use a test not only developed by CDC, but CDC has to wrap it up and send it to the lab, and it’s just going to be state labs doing it.”
The effect was that the nation’s labs could only run tests using the CDC’s kits. They couldn’t order their own primers and probes, even if they were identical to the ones inside the CDC kits. And when the CDC’s kits turned out to be flawed, there was no plan B.
By the way, if you want a full list of the various protocols that are listed by the WHO, they can be found here.
Another in-demand approach would look for antibodies to the virus in the blood of patients, a so-called serological test. That’d be useful, because in addition to identifying people with Covid-19, it could tell you if someone was once infected but then recovered. “The better your surveillance, the more cases you’re going to catch, but even with perfect surveillance you won’t catch everything,” says Martin Hibberd, an infectious disease researcher at the London School of Hygiene and Tropical Medicine who helped develop one of the first tests for the coronavirus SARS in the early 2000s. “Until we’ve got a full test of this type of assay, we don’t know how many cases we’ve missed.”
A serological test would also probably be cheaper than a PCR-based one, and more suited to automation and high-throughput testing. A researcher in Singapore is testing one now.
Here’s an early paper on the topic, if you are interested.
Serological assays are of critical importance to determine seroprevalence in a given
population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of Coronavirus SARS-Cov-2 antibody titers will also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.
As far as I can tell, this method has not been deployed at all thus far, and that applies to India as well. Here’s a Wikipedia article about the different methods of detecting Covid-19 – it’s about more than that, the first section applies here. Here’s an article from Science about a potential breakthrough.
But whether you use any variant of the RT-PCR or the serological test, given the sheer number of kits required, there is going to be crazy high demand, and a massive supply chain problem.
Along with, what else, politics, and bureaucracy:
The Wired article is based on reporting in the US, obviously, but there are important lessons to be learned here for all countries, including India.
Here are some links about where India stands in this regard:
- There may well be indigenous test kits that cost a lot less (fingers crossed!)
- The last two questions in this article speak to the issue that we have been speaking about here.
- An article from Mashable about this issue.
- Scroll on whether we are testing enough.
I’ll be updating the blog at a higher frequency for the time being – certainly more than once a day. Also (duh) all posts will be about the coronavirus for the foreseeable future.
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Thanks for reading!