A pandemic was something I never thought I’d experience in my lifetime. As of this writing, COVID-19 has infected at least 471,417 thousand people (a lot are still probably undetected), causing 21,295 deaths, affecting 170 countries. (Good news: 114,642 have recovered.)
COVID-19 is an infectious, respiratory disease caused by SARS-CoV-2. It has symptoms such as cough, fever, tiredness, and difficulty of breathing.
WHO recommends aggressive testing and contact tracing to fight this pandemic. COVID-19 testing has been a controversial topic recently — we’ve heard of RT-PCR everywhere. But how does the testing really work?
Here’s a simplified guide to testing. I admit a lot of these things are really technical but I tried to make this as simple as possible, inserting key molecular biology concepts every now and then. I’m not an expert myself, but this is a compilation of my readings, experience, and theoretical knowledge. Let’s learn something new today ✨
We’ll break it down into FIVE simple concepts:
- Sampling & Extraction
- Primer & Targets
But before we start, take time to learn the central dogma of molecular biology and genes first. Move on when you feel you have an idea of the concept 🧬
Assuming you understood the central dogma, let’s move on to the testing. It starts with sampling – following the DOH protocol on who we have to test.
- Sampling and Extraction
Our goal is to collect viral samples and to isolate RNA.
For COVID19 testing, the RNA is isolated from upper and lower respiratory specimens (e.g. nasopharyngeal swabs).
What we do is we get the RNA from the sample using an RNA extraction protocol that scientists have developed. Since we collected everything present in that area, our extraction can contain the RNA of your own cells, bacteria, and virus.
2. Primer and targets
This gets trickier! With all the RNA there, how do we detect the SARS-CoV-2 RNA?
To understand this, we need to discuss two concepts:
When scientists design primers, they try they best to make sure that the PRIMER only finds that “snippet” which is only found in their target.
Overwhelming? Let’s take this slowly.
So what are these “snippets” or paragraphs we are looking for in the SARS-CoV-2
The common target is the nucleocapsid gene which gives instructions for how the nucleocapsid protein is made in the virus.
Now, we discuss why we use real time RT PCR or reverse transcription polymerase chain reaction, which is a technique that combines reverse transcription and amplification of gene target. In a simplified overview, we run the primer + patient’s sample in this machine to see if the sample has this “snippet” indicating the presence of the target (virus).
WOAAAH wait, don’t get overwhelmed. Let’s break this down to three important concepts:
- Reverse transcription
- Real time
Let’s incorporate these concepts into the central dogma.
Stay with me here. This is the most crucial part for the laboratory diagnosis of COVID-19.
To say that the TARGET (in this case, the SARS-CoV-2 nucleoprotein RNA) is present in the SAMPLE (nasopharyngeal swab), the count must reach a certain threshold.
Analogy: I want to harvest 100 fruits (threshold), and my trees bear fruits at the same speed (replication by PCR). I will reach 100 fruits faster if I start with 21 trees (more viral load), than if I start with 3 trees (less viral load). And, if I have no trees (virus not present; covid-), I will definitely not reach the threshold. The real time RT-PCR helps us count the number of trees (virus) we started with.
To look at it another way, it will take more time for me to reach 100 if I start with 3 trees than if I start with 21 trees. And in RT-PCR, we set the “time.” Thus, once the time is up, only the ones that reached the threshold will be considered positive.
Of course, this is more complex than this. Again, I am only simplifying it so the concept will be easier to understand.
To reiterate, if it reaches the threshold, then I’m quite confident that I have the virus.
Lastly, we go to errors.
The reason why we are not fully confident is because no test is perfect. What do we mean with errors? See the different scenarios below:
False positives are also possible but we focus on the false negatives because there are serious risks to the patients and to others. For example, if I tested negative when in fact I have the disease, I might not be able to take precautions to isolate myself from my family — endangering them for possible infection as well. That is why negative results must be combined with clinical observations, patient history, and epidemiological information.
What are the reasons for having false negatives?
Before I end this, I just want to reiterate the urgent need for testing.
Lastly, I’m calling on the government to focus their solutions to improving our citizen’s health and wellbeing (health is multifactorial) and strengthening our health workers and research sectors, instead of running around like headless chickens and sounding like clanging cymbals. Make sure no one is left behind. Have better contingency plans, risk assessment, and most importantly, EMPATHY. Solutions should involve everyone not just a certain few. We subject you to the highest standards and you must deliver because Filipinos deserve no less.
And with that, I end this mini mol bio lesson. Thank you for learning with me today! I’m preparing a Filipino version of this as well. I hope I finish it soon. If you have questions, suggestions, comments, just message me anytime! ☺️
This too shall pass.
Download the file here: bit.ly/SimpleCOVIDGuideEng
- Centers for Disease Control and Prevention (2020). Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19). Retrieved on March 2020 from https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-specimens.html
- CerTest Biotec. (2020). Viasure Real Time PCR Detection Kits.
- FDA. (2020). Fact Sheet For Healthcare Providers: CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Retrieved on March 2020 from https://www.fda.gov/media/134920/download
- FDA. (2020). LabCorp COVID-19 RT-PCR Test EUA Summary. Retrieved on March 2020 from https://www.fda.gov/media/136151/download
- Hao, Q., Wu, H., Wang, Q., et al. (2020). Difficulties in False Negative Diagnosis of Coronavirus Disease 2019: A Case Report. Research Square. doi 10.21203/rs.3.rs-17319/v1
- Ortiz-Ospina, E., & Hasell, J. (2020). How many tests for COVID-19 are being performed around the world? Retrieved on March 2020 from https://ourworldindata.org/covid-testing
- World Health Organization. (2020). Interim Guidance for Laboratory testing for coronavirus disease (COVID-19) in suspected human cases. Retrieved on March 2020 from https://www.who.int/publications-detail/laboratory-testing-for-2019-novel-coronavirus-in-suspected-human-cases-20200117
- World Health Organization. (2020). Q&A on coronaviruses (COVID-19). Retrieved March 2020 from https://www.who.int/news-room/q-a-detail/q-a-coronaviruses#:~:text=symptoms
- World Health Organization. (2020). WHO Director-General’s opening remarks at the media briefing on COVID-19 – 16 March 2020.Retrieved on March 2020 from https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19—16-march-2020
- Worldometer. (2020). COVID-19 Coronavirus Pandemic. Retrieved from https://www.worldometers.info/coronavirus/ (Last Updated: March 26, 2020, 04:09 GMT)