This post has been reviewed by one of our faculty experts, Dr. Scott Covey of UBC Biochemistry
Image credit: “An image of the CDC’s COVID-19 diagnostic panel” by Centers for Disease Control and Prevention is in the public domain
Wikipedia: https://zh.wikipedia.org/wiki/File:CDC_COVID-19_test_kit.jpg,
The paper that we’ll be demystifying can be found here, if you want to follow along.
By this time, you’re likely aware of the swab test: a nasal swab administered to determine if someone has been infected with SARS-CoV-2. However, that’s not the whole story when it comes to testing. After all, if it was that simple, people wouldn’t have to wait so long for the results.
Once the swabs or other sample reach the lab, they’re analyzed by a real-time reverse-transcription polymerase chain reaction assay, or real-time RT-PCR.
Let’s break that down.
In organisms like ourselves, DNA contains all the instructions we need to function in the form of molecular bases. Our cells then read these instructions, creating a copy of them in a similar molecule named RNA, which can be used to direct the production of proteins and enzymes that keep the body running. This process of reading and creating an RNA copy is called transcription. Viruses like SARS-CoV-2 don’t have DNA, instead starting off with RNA, invading our cells and letting us do the heavy lifting of making their proteins.
“RT,” or reverse transcription, generates fragments of DNA from this viral RNA. These pieces of DNA can be traced back to the virus that created it, be it SARS-CoV-2, influenza, or HIV.
“PCR,” or the polymerase chain reaction, makes billions of copies of the DNA fragments created by reverse transcription. After all, a single molecule of DNA can be difficult to work with. PCR mimics our own cell’s ability to copy our DNA using cycles of high temperatures and enzymes.

“Real-time” describes how the amounts of each type of DNA fragments can be monitored as this process occurs. Primers let us know exactly what type of DNA fragments are being generated, helping with SARS-CoV-2 identification.
Now, our existing real-time RT-PCR methods are working well enough, but they’re not perfect, with room for improvement in certain areas. Higher sensitivity, for example, would allow doctors to zero in on even smaller amounts of viruses than the existing test would miss. Higher specificity could prevent other, similar viruses like SARS-CoV from sounding off a false positive.
Lucky for us, a team from the University of Hong Kong have devised a new test to improve these aspects of real-time RT-PCR testing for SARS-CoV-2. Their test looks for two very distinct regions in SARS-CoV-2, the RNA-dependent RNA polymerase (RdRp), an enzyme that copies RNA, and a helicase (Hel), an enzyme that separates the two resulting strands of RNA. As a result, they called it the RdRp/Hel real-time RT-PCR assay.
The team tried out their new test on samples from Hong Kong patients with either COVID-19 or an entirely different viral respiratory infection, the test result already being known. Additionally, they began with three new tests, the other two looking for RNA corresponding to the nucleocapsid, which contains the virus’ RNA, and RNA corresponding to the spike, which plays a role in infection. These two were eventually weeded out as candidates.
The RdRp/Hel assay managed to score more positive reads for SARS-CoV-2 than the current test in both lab-generated samples and clinical patient samples. Specifically, the RdRp/Hel assay shows higher sensitivity in not only nasal and throat swabs, but also in saliva and plasma, the liquid component of blood.
The new test also could detect SARS-CoV-2 from samples later in a patient’s course of COVID-19 infection. It found traces of the virus in swabs from Day 12 of infection. In saliva, they could even use it to find traces of SARS-CoV-2 after 18 days of infection.
To test for specificity, the team used 22 swab samples from patients with non-COVID-19 respiratory infections. The new test didn’t give a false-positive on any of them. In contrast, the current test that only checks for RNA from the RdRp, was found by the team to react with SARS-CoV (not SARS-CoV-2) on two separate trials with three replicates each.
It’s important to note that this novel RdRp/Hel and the current RdRp used different reagents, PCR cycling conditions, and other associated PCR elements, like primers. As such, it is challenging to find out the exact reasons for their different sensitivities.
This new assay has a lot of potential, thanks to these researchers from the University of Hong Kong. And the better the tests we have, the safer we’ll all be.