GenEd's COVID19 Resources
GenEd is here to help your small lab, hospital, or clinic increase COVID19 testing capacity. We are currently offering CDC-recipe viral transport media (VTM) for sale. In addition, we freely offer our strategy for validating a real-time PCR assay for the SARS-COV2 RNA genome. We are here to help, please contact us with any other questions.
We're going to illustrate what we would do if we were hired to validate a COVID19 assay for an independent clinical diagnostic lab. Let's check our assumptions before we start. We assume the client lab has:
1) CAP/COLA accreditation.
2) access to the equipment necessary to carry out the reaction.
3) access to a certified/licensed technician who can work independently in a "molecular" setting.
After verifying the client lab is accredited and has the right technical infrastructure, we would arrange a phone or virtual meeting with lab staff to discuss the FDA EUA. COVID19 assay validation, for the time being, is not like a typical molecular validation. In normal times, an accredited lab is allowed to roll out a new test using the regulatory mechanism of the LDT or Laboratory Derived Test. These are, of course, not normal times. Because of the public health impact of unregulated testing for a pandemic virus, FDA and CDC are usually in control of all testing through the state public health labs. FDA allows independent clinical diagnostic labs to participate in COVID19 testing using an EUA or Emergency Use Authorization mechanism.
FDA highlighted the parts of the EUA that need to be completed by the client lab. We're not going to discuss obvious things like the lab's name and address, name of the machines used etc. These are things we would expect the client lab to want to fill out on their own for cost savings.
We'll discuss the parts of the EUA where labs need help. The EUA asks for a focused device description and test principle (part F),
and then a limited susbet of typical validation experiments (part H).
Let's look at each EUA section in detail:
The EUA gives the client lab the option to attach the laboratory procedure as an appendix to satisfy the Description. We recommend this option. Having the lab's COVID19 testing procedure reduced to a procedure should be done as part of the validation anyway. Using a written procedure in section F-2 enforces good compliance discipline while simultaneously focusing the technical staff. Note that the CDC provides a very serviceable SOP.
In the early days of COVID19 testing, finding positive controls was the rate limiting step in assay validation. Thankfully there are now multiple sources of control RNA. We will not recommend a supplier, but will recommend quality metrics upon request. BEWARE A TEST SUPPLIER WHO THINKS RNaseP IS A GOOD CONTROL FOR THE REVERSE TRANSCRIPTASE STEP! The RNaseP gene, the "standard candle" for copy number reference in almost all human molecular diagnostic testing, is indeed expressed as RNA and should be seen in COVID19 testing specimens. Some test suppliers think this is a good enough control for the reverse transcriptase step that converts the COVID19 genome from RNA to PCR template. We thouroughly disagree. Even small amounts of RNaseP gDNA in an imperfect RNA extraction prep will be enough to show a positive RNaseP signal, potentially masking dead reverse transcriptase enzyme in combined master mixes.
It is not possible to distinguish dead reverse transcriptase from a COVID19 negative result unless there is at minimum an external RNA positive control. Because a false negative COVID19 results may lead a COVID19 carrier and their healthcare provider to making decisions that affect at-risk populations, we recommend strigent controls for the assay.
What does this mean, in concrete assay design terms? It means every COVID19 test run should have, at minimum:
-a well with either a synthetic RNA construct or inactivated virions in place of extracted specimen.
-a well with the typical no template control; preferably the same nuclease-free water used to prepare the reaction before aliquotting.
Depending on which primer/probe sets the client lab chooses, an internal (per well) control would consist of RNA from the conserved N protein region shared between all SARS-type coronaviruses. This would show that each well had the right conditions to reverse transcribe a similar RNA sequence and PCR amplify that target in the real-time PCR assay. If RNA control is still limiting, it is less optimal but still technically acceptable to use only an external positive control. ONLY IF all reaction components are aliquotted from the same tube before specimen is added. The EUA specifically asks for the client lab to state the frequency that controls will be performed. The answer is at least one external positive control well (synthetic or inactivated COVID19 RNA) per run and, if reagent availability allows, a non-COVID19 RNA per well as an internal positive control.
The answer here depends on the primer/probe sets used. The Chinese CDC uses a single primer/probe set in ORF1ab and another in the homologous region shared by all SARS-type coronaviruses (N protein). The US CDC assay, which seems to be what most American suppliers are copying to fit under the EUA, targets two regions in the COVID19 N gene and RNaseP. Since most labs in our audience will be using an EUA-compliant assay supplied by a commercial developer, it is best to carefully follow the guidelines in the CDC SOP on pgs 31-32
We'll quote directly from the EUA here:
Laboratories should document the limit of detection (LoD) of their SARS-CoV-2 assay. It is acceptable to spike RNA or inactivated virus into artificial or real clinical matrix (e.g., BAL fluid, sputum, etc.) for LoD determination. It is recommended that laboratories should test a 2-3 fold dilution series of three extraction replicates per concentration, and then confirm the final concentration with 20 replicates. FDA defines LoD as the lowest concentration at which 19/20 replicates are positive. If multiple clinical matrices are intended for clinical testing, laboratories should submit the results from the most challenging clinical matrix to FDA. For example, if testing respiratory specimens (e.g., sputum, BAL, nasopharyngeal (NP) swabs, etc.), only results from sputum need to be reported in the EUA application. If needed, we recommend that you follow the most current version of the CLSI standard, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures (CLSI EP17)
The real art here comes in determining what concentration range to use for the 2-3 fold dilution. We are cautiously recommending a middle value of 200 copies/uL in a final reaction volume of 10 to 20 uL. Depending on the master mix used, low and high values of 20 or 500 copies/uL should give acceptable Ct values. Please note that LOD determinations are very dependent on technician skill.
Let's quote the EUA again:
It is acceptable to conduct an in silico analysis of published SARS-CoV-2 sequences using the assay’s primers and probes. FDA anticipates that 100% of published SAR-CoV-2 sequences will be detectable with the selected primers and probes.
How does one do an in silico analysis of published SARS-CoV-2 sequences? Your tax dollars funneled through NIH have made this surprisingly easy. Go to Primer-BLAST:
1) Enter in the forward and reverse primers in the "Use my own . . ." fields.
2) Change the "# of primers to return" field to 1000.
3) In the Primer Pair Specificity Checking Parameters block, change the "Database" field to nr.
4) In the Primer Pair Specificity Checking Parameters block, change the "Organism" field to blank
5) Click on the "Get Primers" button and make yourself a cup of coffee or stretch while waiting.
If using any of the government designed primer pairs, you can expect to see the bat coronavirus sequence and lots of different isolates of COVID19. Cut and paste these into a document and attach them to your EUA.
What about the probe sequence? We've got another freely available searchable database solution for that. Go to the venerable BLAST.
1) Cut and paste the probe sequence into the "Enter Query Sequence" field.
2) Change the "Program Selection" radio button to Somewhat similar sequences (blastn).
3) Click on the "BLAST" button and wait for results.
Don't be surprised if you get lots of different primates in your results for the RNaseP probe sequence. RNaseP is highly conserved across primates and is expected to give BLAST hits in Hylobates etc. Cut and paste your results into a document and append to your EUA.
The EUA gives labs the option of an in silico analysis OR actual experiments to show that the assays primer/probe sequences don't target genes in other organisms commonly encountered in the respiratory system. A clever validator could make the case that the in silico results of H-2 could sub in here. As stated in this section of the EUA, FDA considers predicted homology of greater than 80% as evidence of cross-reactivity. If the Primer-BLAST results, set to look for 1000 targets, didn't return a sequence with > 80% homology in a species listed in the table in this part of the EUA, chances are the primer pairs are specific. Alternatively, a hyperspecific in silico analysis can be performed by following the steps above for BLAST searching, while setting the "Organism" field to the species being tested. While an in silico analysis here can make the EUA faster to finish, we recommend that labs actually add in purchased genomic DNA from representative species in the EUA's table. It's good practice in assay setup and confirms that the lab can rule out possible contaminants for inconclusive results.
Quoth the EUA:
In the absence of known positive samples available for testing, laboratories should confirm the performance of their assay with a series of contrived clinical specimens by testing a minimum of 30 contrived reactive specimens and 30 non-reactive specimens in a randomized blinded fashion. Contrived reactive specimens can be created by spiking RNA or inactivated virus into leftover individual clinical specimens representing unique patients; the majority of these specimens can be leftover respiratory specimens such as NP swabs, sputum, etc. Twenty of the contrived clinical specimens should be spiked at a concentration of 1x-2x LoD, with the remainder of specimens spanning the assay testing range. FDA defines the acceptance criteria for the performance as 95% agreement at 1x-2x LoD, and 100% agreement at all other concentrations and for negative specimens.
We recommend labs follow this advice to the letter. Any labs interested in plate maps for this part of the validation should contact us!
COVID19 Testing Resources
Here are helpful links to sources of information and tools we use for assay design and validation planning. A link from GenEd does not imply endorsement of any company's products or services and are strictly for informational purposes.