March 6, 2020

What is the Coronavirus PCR Test?

My PCR primer-probe sets, order for up to 50 tests that detect the genetic sequence of the COVID19 virus. Ordered on March 1, 2020, online on a Sunday. They arrived in my lab on March 3rd, Tuesday morning just after 10AM. Wednesday, I designed a test protocol for reaction set up with my technologist assistant, Ed Guzman, while I completed some committee work that day and worked on our VEGF and Norrin research. Thursday, March 5th, Ed set up reactions to run, which were completed in a few hours by the end of Thursday. This is not an FDA approved diagnostic test, but it is a test using primer probe sets ordered from the manufacturer that makes the same probe-sets for test kits in use. My cost for these COVID19 specific components was $170 US. We used New England Biolabs (USA) 2x real-time PCR (Probe) Master Mix, and an Agilent AriaMx Real-Time Thermocycler for our reactions. 
What is the coronavirus "PCR" test as recommended by the Centers for Disease Control (CDC) and also used by other countries around the world? One of our jobs as a University scientist is to make sure you understand how things work, like the coronavirus test. What is it, and how does it work?

I am a molecular biologist, a biochemist, who does biomedical research and it turns out that we use the same method, "Real-Time PCR", to measure the expression of genes we study in the retina and in retinal-vascular diseases. So we have made some videos to show you how the test works using the same CDC recommended PCR primer/probe sets. They are on YouTube to make them easy to view. What did I learn? Mostly that there is no excuse for the fact that the country that put the first men on the Moon did not have COVID19 testing ready. I ordered PCR reagents from the same company that makes primer-probe sets for the CDC on a Sunday, had them arrive on a Tuesday. We took one day to prepare the first test PCR reactions and run them. 

My playlist on YouTube has videos related to this demonstration, starting with the first video where our PCR reagents have just arrived in our lab. 

One should understand a few points about the coronavirus PCR test. First, a new PCR designed to detect a new target RNA/DNA sequence does not necessarily work well in PCR reactions until it is actually tested at the lab bench. Most diagnostic tests for medical labs are developed with far less urgency and more time, and they are often easier to automate. Real time PCR based analysis is not so cut an dry as some tests and a well trained technologist or full scientist must review the PCR reactions and understand how positive and negative controls have worked (or not) to interpret the final result. PCR and real-time PCR is mostly used for research purposes. We use it to measure the activity of any gene we study, but if we need to fine tune the PCR design over several days, or even weeks, that is ok because it is research not medical diagnosis. 

Many factors can alter if a PCR reaction works as desired or fails to work. There are many makers of enzyme mixes and reactions solutions, and there are also many different brands of real-time PCR instruments. A particular PCR-based test may run well with one brand of enzyme mixes but poorly with another. The CDC's FDA-approved test uses PCR primer sequences that a molecular biologist can order like myself, but the FDA-approved test is specific for only one or a few brands of reagent sets, and even approved for use in a limited number of Real-time PCR instruments. That is standard practice to have a diagnostic test that must be used the same way in all labs. Now you can imagine that this takes time to establish such a protocol. It even takes weeks. However, when the "bombs are already falling", sometimes you need to go for speed and get testing.

You do not need the PCR-test to have a doctor treat you for your symptoms, but the test is extremely useful to predict how a pandemic disease is spreading geographically, in each country and in each State. It is like having data on pressure and temperature in different locations for meteorologists to run weather models that can predict good or bad weather in the future. For the United States, doing thousands of tests for COVID19 many weeks ago would have helped to get data for forecasting COVID19 spread in the USA. We may never get to that prediction tool and benefit from deploying resources to anticipate need because we have missed the boat and COVID19 will spread rather quickly in some locations around the country. While some countries like South Korea are testing over 10,000 persons per day, the US had only tested about 6,000 people total at the first writing of this note.

Despite all of this, I want you to know what this PCR test is that you hear about all over the news and social media. First, so it is not a strange black box, you should have real knowledge about the coronavirus PCR test. Basically, how it works. What kinds of machines are needed in the lab to do the test. If you see things online that offer tests and want to charge you, they are bogus, or home test kits are bogus too. The PCR reactions must be run in a PCR instrument. They also need to be run in a REAL-TIME PCR instrument, a special type of PCR machine that allows us to see the reaction unfold in real-time. 

To demonstrate how the test works, I ordered these reagents, called PCR primer/probe sets, from a commonly used manufacturer of such PCR reagents (IDT-DNA) that has been providing these reagents around the world for weeks now. I also obtained from them some small circular "plasmid DNAs," which are ways that we biochemists use to hold DNA sequences of interest. These ones have small portions of the coronavirus RNA/DNA sequence which are detected by the PCR tests. These plasmids are used routinely so labs can design PCR-based tests for these viruses. They are not viruses and are not harmful, but one needs to have a positive control to simulate a sample from someone who is sick with the actual virus. This would alert us to the fact that the reaction may have failed to work as desired and so would need to be repeated. 

I have made four videos, short, so far. If you want to understand what the PCR is first, then watch Video 2. PCR stands for Polymerase Chain Reaction, and that is a method to take one copy of a DNA sequence and make millions of copies very quickly. 

While I have used the same primer/probe set sequences as recommended and in the kits assembled by the CDC, I am not using the CDC's kit and I am using a particular brand of PCR enzyme reaction mix that I have found is very reliable and sensitive in my research. If we got to the point where much more testing was needed in a dire emergency, the country could utilize research labs but we would have to fall outside the zone of officially approved FDA tests and I do not think we will need to resort to that for treating people. People will arrive at ERs and they will just need treatment as soon as possible. However, it is important for you to appreciate that the people with skills to run these tests for us and our loved ones are able to do so by getting educated about biochemistry and molecular biology in our colleges and universities. Many of the PCR primers used to test for this virus around the world were actually designed in academic virus-research labs and in institutes like the CDC. 

You can see all four videos in a playlist here:

Corona Virus PCR Test: How it works video playlist at YouTube

Video 1: See what the reagents look like, just four small tubes.

Video 2: I draw out how the test works and how PCR works.

Video 3: We meet Ed, one of my assistants who runs these kinds of analysis reactions and who sets up the test in our real-time PCR instrument.

Video 4: The data from our first run on the instrument happening in real-time. It looks like the CDC recommended PCR primers work just fine.

Ken Mitton
Remember, you can be your own scientist too, as long as you think for yourself :) and you can.

PS. If anyone tries to make an excuse for any western nation's failure to have PCR testing ready to go, you should know there is no excuse. The knowledge was present, the skilled scientists and technicians were present, the suppliers of specialized chemicals required were present. The failure is where the buck stops, at the top. Period.  

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