How to Validate a COVID-19 Assay

The FDA is now democratizing the testing of the novel coronavirus: SARS-CoV-2 (the virus which causes the COVID-19 disease syndrome—I will call it COVID-19 from here on as that is the colloquial name most people know) by allowing high complexity testing labs across the United States. This move will permit more labs to test for COVID-19. A previous post by contributor Constantine Kanakis describes the biology of the virus, so I will not repeat that material. Instead, I will focus on some considerations in validating a Lab Developed Test (LDT) COVID-19 molecular assay.

The president of AACC, Carmen Wiley, said there are 11,000 high complexity testing labs in the US, which could qualify for performing this testing. However, not all of these labs have molecular and virology expertise, so others have placed the number of labs with qualified staff and instrumentation at 400.

Published Assays and Targets: As an overview, the figure below (Figure 1) summarizes some published COVID-19 assays. As you can see, the major strategy involves using the TaqMan probe strategy where a short probe is degraded by Taq polymerase releasing a fluorescent molecule (green ball) from a quencher molecule (blue ball). The TaqMan approach allows for quick performance of the assay and easy interpretation. One lab from Japan is using nested PCR amplification and sequencing of the Orf1a and S genes as well.

Figure 1. The COVID-19 genetic structure is abbreviated above with the different genes targeted displayed. The names of institutions that have published their assay procedure along with the TaqMan reagents that were reportedly used with each assay are shown above. Primers are represented by small arrows with a TaqMan probe in the middle represented by a black line with green and blue circles indicative of the fluorescent molecule and its quencher. The double set of arrows for the Japanese assay represents a nested PCR strategy.

In silico Cross-reactivity:

The FDA guidance allows cross-reactivity to be minimally assessed in silico by demonstrating “greater than 80% homology between primer/probes and any sequence present in the targeted microorganism.” The primer locations can be found in the publication of each protocol (except Thermo) and can be confirmed by checking the NCBI Blast site and they actually have a selection for beta-cornavirus (Figure 2) now that allows you to search for your primer’s reactivity across other related viruses- Very helpful!

Figure 2. Select Betacornavirus before entering your primer/probe sequence to confirm cross-reactivity.

Primer/Probe Design:

The N region is the most popular site to probe and is included in most kits once and the CDC kit three times. It was the reagent set for N3 in the CDC kit that was having difficulties, so you may decide to not include that component in your LDT. If you want to see how the different available primer sets align on the N gene sequence you can see below for the primers labeled based on their source. Many are overlapping, perhaps because many people thought the same site was a good target (Figure 3).

Figure 3. N-gene of COVID-19 along with labeled primers from some published assays. The information on the source of the sequence is shown on the bottom right with the link.

Commercially Available Assays:

An important part of validating your COVID-19 assay is to do so quickly. Thus commercially available kits would be helpful, however there are only two commercially available sources at this time: IDT and Thermo. IDT is producing a kit with the CDC design. Thermo produced their kit over the last few months and does not have any published validation information that I could find. Also Thermo when I checked just now for the catalog number, it says this product is unavailable… not sure what that means, but maybe you can try contacting them. Both IDT and Thermo list control plasmid reagents for their assays.

Controls for the Assay:

The wording of the FDA announcement was interesting in that it 1) did not require clinical samples, but allows “contrived clinical specimens.” “Contrived reactive specimens can be created by spiking RNA or inactivated virus into leftover clinical specimens.” A major difficulty is the access to actual COVID-19 RNA or inactivated virus. I noticed that the guidance didn’t say that the assay MUST use RNA. Thus most labs would have access to plasmid DNA, which could potentially be used.

Given the limited availability of RNA for validation use, a lab may consider performing much of the assay optimization with COVID-19 Plasmid DNA while waiting for access to RNA. I would like to be sure my assay could extract, amplify and detect RNA as part of the clinical validation.

Asuragen can produce Armored RNA, with synthetic RNA packaged inside of a viral capsid, which would be a useful control for extraction, amplification and detection. However, we heard this will not be available for another month.

Tom Stenzel (director of the Office of In Vitro Diagnostics and Radiological Health at the FDA’s Center for Devices and Radiological Health (CDRH)) said FDA, BARDA, and the CDC will prioritize and coordinate shipments of viral materials to labs when they are ready to validate tests according to a webinar with labs on Monday. Currently, the FDA is directing inquiries to BEI, which is reportedly prioritizing requests to send out samples in 12-72 hours.

Lastly, one could try to use in vitro synthesized RNA sequences surrounding your primer targets as a control for now and may have better luck in getting the product soon. This is the control that is being shipped with the CDC kits to public labs.

Limit of Detection is an unknown for what is likely to be clinically relevant as we don’t know what the levels look like in people with early vs. late vs. severe vs. mild disease. The FDA just says you should be able to detect 95% of samples (19 of 20) that are x1-x2 the limit of detection.

FDA Notification:

This is the final and important step. Once you go live, you must notify the FDA with an Emergency Use Assay (EUA) form within 15 days. Reviewing the form, there doesn’t appear to have complex explanations or overdue requirements for reporting, which wouldn’t be found in a standard lab validation document.

Final Thoughts/Future commercial solutions:

This information is the best of what I know right now based on current information- this is not a complete guide and the FDA guidance should be read closely for all compliance details. Information is changing quickly and is likely to change more if the number of COVID-19 cases in the United States increases. Cepheid, Luminex, and BioFire are reportedly working on assays that will be out in several months and would be easy to use for many labs that already have one or both of these systems-however it may require a full validation for an LDT, but I’m not sure as it is an EUA-further clarification on this point is needed. Although there are several commercial solutions available, we don’t know how demand could impact supply from each company. Fortunately, some large reference labs like LabCorp and Quest are looking to develop a COVID19 test. Good luck, stay safe, and feel free to contact me with any questions in the comments below so that everyone can benefit from the discussion!


In lieu of a list of references, I’ve included web links for the most current and direct sources of information.

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

FDA Guidance to Develop Novel Molecular Diagnostic Tests for SARS-CoV-2

A laboratory advisory from the CDC:

The Food and Drug Administration (FDA) issued new guidance on February 29, 2020, for laboratories to be able to develop novel coronavirus (COVID-19) molecular diagnostics tests and begin use prior to obtaining Emergency Use Authorization (EUA). This permits laboratories that are CLIA certified and meet requirements to perform high complexity testing to start offering severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostic testing after validation is completed as outlined in the guidance. Laboratories should submit an EUA request to the FDA within fifteen business days after validation. FDA will be hosting a webinar to provide more information on March 2, 2020, at 3 pm ET.

Clinical laboratories should contact their state health departments for guidance if they have a suspected COVID-19 case specimen. Clinical laboratories should NOT attempt viral isolation from specimens collected from COVID-19 persons under investigation (PUIs). For interim guidelines for collecting, handling, and testing clinical specimens from PUIs for COVID-19, please see the CDC Coronavirus Disease 2019 (COVID-19) website.

The Crown

Hello again everyone! Welcome back.

No, I’m not talking about Netflix or HRH Queen Elizabeth II, nor am I making references to tiaras, bars, beer brands, or imminently deliverable babies…I am, of course, talking about Coronavirus as it would certainly have caught most of our collective attention in the media by now.

I really enjoyed writing last month’s list of what I think are important things on the horizon for pathology and laboratory medicine this new year, but this month let’s take a more topical turn. So put your surgical masks on, wash your hands, quarantine the next 10 minutes of your time and get ready as I take a shot at the novel 2019 coronavirus outbreak!

***Let’s talk about you and me, Let’s talk about COVID-19…***

Less-Than-Common Cold

A long time ago, in a galaxy far, far away (aka: last year, about 20 minutes north of my apartment in Manhattan) I was in medical school, on rotations on the floors of a hospital in the Bronx. I experienced the surges of two flu-seasons and had a fantastic little mnemonic to remember the viruses that caused colds in most patients. Depending on age and immune system status, you had to think about the principal three viruses we see all the time—I remembered them as: “c-A-r,” note the capital “A.” Let me explain; the letters correspond to coronavirus, adenovirus, and rhinovirus. The are in a general order of when they appear during the months of the year (as coronavirus and rhinovirus kind of switch off in the spring, while adenovirus is around always thus is capital designation). There are a few hundred viruses which contribute to cold/flu-like symptoms in patients and, unless a patient is compromised in some way, we really worry most about one of them. Hint: it’s the one we give shots for annually, more on that in a minute.

Image 1. Okay this is a little better than my seasonal cold “cAr”…Seasonal variations in frequency of selected upper respiratory tract infection pathogens. Funny that, of all the colors, Influenza gets the good ‘ol fashioned “red alert” from the commanders on the USS Medscape. PIV = parainfluenza virus; RSV = respiratory syncytial virus; MPV = metapneumovirus; Group A Strep = group A streptococcus. (Source: Medscape)

As far as this coronavirus outbreak is concerned, this is a “novel” (i.e. new) variant (read: mutation) of a respiratory viral pathogen that is affecting a disproportionate number of patients in higher severity than expected. Its official entity name has now been filed by the World Health Organization (WHO) as COVID-19—corona virus disease of 2019. The actual virus is a relative of the infamous SARS virus from the early 2000s. That was SARS, this is SARS 2.0—literally. This virus is designated SARS-CoV-2. SARS stands for Severe Acute Respiratory Syndrome and is caused by strains of coronavirus found in the remnants of infected individuals’ coughs and sneezes—please wash your hands—and causes a spectrum of symptoms from mild to severe including pneumonia, respiratory disease, and even renal failure.

Image 2. Heeeerrreee’s COVID, well at least a coronavirus model. Named after the crown-like spikes on its envelope surface which act as receptor anchors in order to attach to cells and infect us. The receptors’ favorite target? ACE2 (angiotensin converting enzyme 2) receptors—am I getting your blood pressure up yet? I said renal failure…full circle, right? (Source: Centers for Disease Control and Prevention)

How Does this Even Happen?

Okay, who took a sabbatical to Wuhan, China, and ate a wild fruit-bat salad? No one, that’s not how this works. But, if you’re looking for quick grocery store recommendations at the present moment I’d probably tell you to check out ALDI or a farmer’s market a few spots higher on the list than the Huanan Seafood Wholesale Market in Wuhan which harbored a majority of outbreak case-cause tracings. The bottom line is that COVID-19 and the SARS-CoV-2 have appeared in the world the same way the previous similar outbreaks have—through zoonotic mutations which then spread to humans. This zoonotic transmission is so effective to presenting humans with super infectious entities because it sends us pathogenic material we would have never seen before and our “naïve” immune systems are caught off guard. Now don’t get all panicky; yes, I’ve seen Contagion, Outbreak, and read The Andromeda Strain—in fact, I absolutely love when epidemiological medicine has the media spotlight. It’s a very exciting way to showcase public health, medicine, and—our favorite—laboratory professional work!

Image 3. Okay, no bat salad but this outbreak does involve bats endemic to the regions in question. It also involves, pangolins, kind of like armadillos, which are bought and sold (and trafficked) all around the world for various reasons. The pangolin DNA matches the SARS-CoV-2 genome highly, so scientists assert that its involvement is inherent. The complications in sanitation and animal/food handling are obviously implicated in this and other outbreaks, but dealing with COVID-19 requires a strong public health response. (Source: Osmosis)

Basically, this process of mutation and transmission is the modus operendi of a viral particle. You can’t quite kill them, they’re not quite alive by biological definitions, they’re just packaged proteins on autopilot. They’re kind of like natural robots that want to propagate their species by adapting over time—they’re The Borg or Cybermen, depending on your sci-fi preferences. But both offending automaton predators have a mutual enemy in public health—a doctor (get it? TARDIS pilot and/or Beverly Crusher both work wonders in a pinch…) Anyway, it’s never just physicians, but a whole hard-working team of health advocates that conduct surveillance, field research, epidemiologic studies, and first-hand treatment.

***Side note: if you’re bored, in a hurry, or just don’t like my articles—don’t fret! Go watch that Osmosis video on COVID-19 and you’ll be up to snuff on the current outbreak in no time. Or in 12ish minutes.***

***Hey, you made it this far. Great! Interested to know more about the COVID-19 virus from our very own American Journal of Clinical Pathology? Visit here to learn more about the story of how this pesky coronavirus mutated its way into headlines. Fresh off the AJCP presses this month!***

You Should Update Your Antivirus Software

Image 4. Fake news is the new normal. It’s hard to sift through a sea of garbage. You can’t just ignore it, because sometimes there’s serious situations like war—or viral pandemics! So, you’ve got to update your personal mental anti-spam filter and know how to sniff out suspicious sources. Let’s call it “information hygiene” to be consistent with the zoonotic exposure narrative, yea? (Image source: NPR)
Image 5. Let’s lighten the mood a bit. These situations can drive folks absolutely nuts. People obsess over minutiae and often wrong details when archetypal preventions like hand-washing and isolation are paramount to prevent epidemics. Public health and pandemic situations breed conspiracy thinking—wait, never mind, this is a photo of me explaining the residency match process to my friends and family. Kidding. Kind of. (Source: a popular meme, but originally from It’s Always Sunny in Philadelphia)

No doubt in my mind you’ve probably seen plenty of coverage about SARS-CoV-2 in the media. I’d also be willing to bet a lot of it is either dilute, sensational, or possibly even misleading. Regardless, there are always going to be people that don’t “buy in” to the public health message. If you remember Contagion¸ Jude Law’s character pushes the efficacy of “forsythia,” a homeopathic herb supplement that supposedly mitigates the horrible disease spread from southeastern Asia from improper food handling—if I recall correctly, it was a paramyxovirus that time. In this SARS-CoV-2 epidemic we have no current effective treatments, so prevention is key.

In an effort to address this type of health misinformation the WHO and CDC are actively disseminating as much educational information and graphics as they can write. Trying to dispense advice for the public including proper mask wearing, education videos, and myth-busting (i.e. hand dryers do NOT kill the COVID-19 virus, UV lamps do NOT kill the virus, thermal readers are effective in screening populations for symptoms within limitations, alcohol and chlorine do NOT kill the virus, receiving packages from China is still safe, pets don’t harbor the virus at this time, other vaccines do not affect this virus, saline nose sprays do not affect this virus, garlic/oils/other supplements have no effect on this virus, and all age groups are affected)—good stuff there. The most trusted sources of information regarding epidemics should be the representatives of functional medicine and health outcomes, doing work every day to make people healthier. Often times, politics, misinformation, or complex situations make information delivery harder than you’d think and the risks are increasingly high.

Image 6. BONUS MATERIAL UNLOCKED: Glad you made it this far. I mentioned Netflix’s The Crown above, but if you’re looking for a fantastic series that breaks down public health epidemiology, influenza burden, research and surveillance on emerging pathogens as updated as 2019, check out Pandemic. 5/5 surgical masks would binge again. (Source: Netflix)

A Crown of Thorns: Don’t Forget About the FLU!

Flu vaccine deniers: turn away now or be healed! —or at least exposed to another point of view rooted in evidence-only concepts in medicine and population health. Consider the following: as of this month, COVID-19 has infected 43,000 people and killed 1,000 (approximately 2-3%). Remember SARS? That infected 8,000 and killed 700 (approximately 10%). MERS? 2,500 infected, 860 deaths (approximately 34%). And what about Ebola? 29,000 infectious cases with 11,000 deaths (approximately 40%). That was sourced from the Osmosis video with data from the WHO. Pretty impressive right? Well, not if you look at this: according to the CDC, the 2019-2020 influenza burden statistics include 36,000,000 infectious cases, with 17,000,000 clinical visits, 440,000 hospitalizations, and 36,000 deaths. One might say “hey, Dr. Kanakis, slow down there you’re spitting out all these numbers and the facts won’t lie. Looks like influenza only killed 0.1% of cases.” And you know what, you’re right. 0.1% is lower than the other viral epidemics. But check this out, because of the sheer number of cases, that means more people died of influenza than COVID-19, SARS, MERS, and Ebola COMBINED and those happened in other years. That‘s just this year’s flu season alone. I’ve talked before about recognizing and detecting the common cold vs. influenza before, check it out if you’d like a refresher!

Image 7. What’s a statistical percentage data point if it’s a relative, a loved one, or yourself? Protect yourself, protect others, and let’s find ways to make proper health knowledge available and acceptable to the masses while promoting excellent healthy behaviors, together! (Image source: New York Post)

We have influenza every single year, and it kills so many more people than we realize. If you want to talk about a terrifying, global viral epidemic, we’ve already got one. And it’s closer than you think. So wash your hands, reduce exposures if you’re sick or immunocompromised, get proper rest, eat well, exercise, read my articles every month, but most importantly—and I cannot stress this enough—get your FLU SHOT!

Thank you so much, see you next time!

Constantine E. Kanakis MD, MSc, MLS (ASCP)CM completed his BS at Loyola University Chicago and his MS at Rush University. He writes about experiences through medical school through the lens of a medical lab scientist with interests in hematopathology, molecular, bioethics, transfusion medicine, and graphic medicine. He is currently a 2020 AP/CP Residency Applicant and actively involved in public health and education, advocating for visibility and advancement of pathology and lab medicine. Follow him on Twitter @CEKanakisMD