RT-qPCR is indeed the common detection method right now. RT (reverse transcriptase) converts RNA->DNA, PCR (polymerase chain reaction) doubles DNA by replicating it over and over in heat cool cycles. To a first approximation, you get 2^n copies of the RNA (it was converted to DNA) where n is the number of heat cool cycles. By adding a fluorescent tracer, you get the q (Quantitative), so you can see how much DNA exists at each cycle. You can then fit a curve and see how much RNA (via its DNA complement) existed in the original sample. It's not actually that slow, you can do this in a couple hours if you have the right setup. The bottleneck is you do need time for the heating and cooling cycles. You also need to extract RNA from the sample first, which could be another bottleneck. I'm not totally clear on the relative cost, a good ELISA would eventually be cheaper, but qPCR itself isn't very expensive. The benefit is that qPCR is extremely sensitive, you WILL see if there is virus in the bloodstream. You can even use swabs and avoid blood altogether. Also, the minute you have a viral genome, you can make a qPCR test, you just need the sequence. That said, if you cleared the infection you will be negative under RT-qPCR.

An ELISA works differently. In this case you would present a part of the viral protein, the patient's antibodies will bind to it, and you then use another antibody (this one is conjugated to some sort of readout method, fluorescent or biochemical) which binds to generic human antibody. Now you have a stack: viral protein <> patient antibody <> readout antibody. You read out the signal provided by your readout, usually some sort of colorimetric thing (see home pregnancy tests, also an ELISA, slightly different configuration though). This works fast, can be manufactured in bulk, BUT requires do you have a protein(s) (in this case the viral protein) that is/are universally recognized by patient antibodies. It's a little trickier to develop. You also need to produce that protein at scale which can take a little time as well. Major benefit: with a good ELISA you WILL see not only whether someone IS infected but whether someone WAS infected for some period after illness regardless of symptoms. This is essentially the only way you'll get a really good number for baseline infection rate. That said, you likely actually need a blood draw.

In short, we need both, we probably could do a lot better than we are doing with qPCR in testing volumes and we could also really use an ELISA.

Source: PhD biochemist, have personally run these assays in various forms.

EDIT: Acronym expansion, source

Maybe one should note, that there are two possible Elisa based strategies. Yes, You can present some viral protein to detect antibodies produced by the body after serum conversion, as you said. This serum conversion takes at least a week to happen and you need a blood sample. On the other hand you can also immobilize antibodies specific to the viral proteins and directly detect the virus. This is possible immediately after infection and fluids like sputum or saliva can be used. Both strategies are under development for quick tests right now.

This is all true, but detecting the viral proteins directly with reasonable sensitivity and specificity is much more difficult. The reason is that there is just a lot less of it to detect. After seroconversion, the amount of antibody against the virus can be very high and relatively easy to check.

+1 the major advantage of RT-qPCR is it's a massively amplified test. Tiny amounts of virus can be detected. When you're detecting a patient's antibodies to the virus, the patient is producing massive amounts of it. That said, after clearing the infection that production will decline, but still should be detectable at least for a while.

Direct detection of viral particles is substantially harder because for N viral particles you basically get O(N) signal (to put it in CS terms). There are some caveats, your readout antibody can generate an exponential signal for example.

You still need to be presenting the right part of the capsid and have the right material. In the direct ELISA scenario, you need 2 antibodies. One to bind the capsid to your surface, the other to bind it again and either have a readout attached directly or be bound by yet another antibody with the readout (likely the latter). Fortunately, viral capsids are repetitive so you might be able to use the same antibody for both. You ALSO need those antibodies to have low cross-reactivity with sputum or whatever other rough sample you have. When you are looking for a patient's antibodies, the patient's immune system has already taken care of that for you. This is all tricky, obviously it's not THAT hard, but it's not trivial.

RT-qPCR is directly a O(2^N) signal. It's extremely sensitive, requires very little assay development, can be made very specific, can work with little sample prep, so it's a much better front-line, first-to-go test than direct ELISA. Still, we'd all love an ELISA, particularly to patient antibodies.

I definitely agree about the superiority of RT-PCR and the drawbacks of direct detection. Nevertheless, seroconversion can take between 5-12 days from symptom onset [1]. Therefore it's important to outline the different ELISA strategies (Thank you for doing so!), when discussing diagnostic tests.

[1] https://doi.org/10.1101/2020.03.05.20030502

> RT-qPCR is directly a O(2^N) signal.

The signal is actually more like N*2^C where C is the number of cycles (usually around 30). So a huge amplification, but still linear in the starting amount, i.e. O(N). That's actually more useful, generally, since ratios of amplified material stay approximately constant during amplification.

Correct, got my math a little off in haste. Constant gets pretty huge which give massive signal, thanks for the correction :)

On a related note, UC Berkeley is looking for volunteers to help them set up a test center using these techniques - https://innovativegenomics.org/covid-19/

Yes, there are of course drawbacks. But the direct detection is applicable at the same stage of infection as a rt-pcr and therefore usefull for immediate disease diagnosis. Indirect tests after seroconversion are the easier alternative, and will probably be used extensively in the aftermath, to get solid epidemiological numbers.

One disadvantage to RT-qPCR I forgot to highlight, and an advantage to direct detection ELISAs (and ELISAs in general) is that ELISAs are relatively robust when fully developed. RT-qPCR requires a lot more gear. See pregnancy tests. Sits on a shelf for ages, all you need to do is pee on it, works reliably, needs no extra equipment.

Are the tests used in [1] (real-time RT-PCR) the same?

The false negative reported several times from the same patient is interesting, but may be more due to testing source (upper respiratory fluid vs. sputum or lower respiratory sources or blood).

[1]: https://wwwnc.cdc.gov/eid/article/26/6/20-0299_article

Yes, the "q" is often used instead of "real-time" to avoid confusion with RT, which can also be "reverse transcriptase". RT in these cases is the enzyme that converts RNA to DNA.

What's the approximate setup and validation time once you have a sequence?

(that is, what's the scale of the fuck up that the machine testing is still being brought online)

I used to set up and run a large plate of qPCR (we didn't use the RT step in our particular use case) in an hour or so. Mind you this was for something like 200 samples. It took 2-3h to run the actual machine. If I was setting up such an assay, I'd order a bunch of different primers (you need DNA to make DNA) and run a bunch of plates with as many samples as I could to see what primers worked best. You'd include a bunch of negative controls, ideally some controls from patients/cultures infected with similar but not identical virus (for example a different coronavirus). To develop an RT-qPCR assay would take a week or two (rough guess), I think it took about a week to develop the WHO assay.

From what I can tell (mostly anecdotal and other data from friends I trust) the CDC assay used bad primers, they were noisy, showing a bunch of false positives. They also used a potentially dodgy fluorescence readout. I used to use the same readout, but our scenario was full of internal controls and the input sample was far more consistent. For human sample data a different fluorescent method that is more reliable should have been used.

Important ways this can be screwed up: it's helpful to use the same machine, the same protocol every time. This is because qPCR is an exponential assay, which means small errors can have big repercussions.

TLDR: Scale of fuckup here is massive. This really isn't that hard, and we could just have used what the WHO/others were using. I have no idea why they decided to go their own way.

more info: https://www.propublica.org/article/cdc-coronavirus-covid-19-...

Five days ago, it was reported that a German biotech company had already produced 4 million tests.[0]

"The tests use what’s called the polymerase chain reaction, a diagnostic method recommended by the WHO that amplifies the virus’s genetic code so it can be detected before the onset of symptoms. The kit comes with two vials: a primer to help detect an infection, and a synthetically engineered piece of the virus, which labs use to produce a surefire positive match to ensure their machines are working correctly. A lab technician combines these ingredients with a patient’s mucus sample—usually from a throat or nasal swab—and results are usually available in a few hours."

Coincidentally, I recently discovered the burgeoning biohacking scene in which many folks are already using affordable PCR thermocyclers in their home labs to do DNA amplification and crispr-cas experiments. I do fungal sequencing for phylogeny myself.

Given the simplicity of the testing process using PCR, why does it seem like the US is moving so slowly with getting tests in peoples hands? Is it the primer or control virus synthesis? Is it the manufacturing process? Is it red tape? If so, which specifically? Is it poor communication? Is it a confluence of factors?

Why isn't every local university or lab working together to at the least, copy the Germans protocol and get to work on manufacturing ASAP? Why isn't there a line of people outside these labs ready to drop-off their sample and get the results online? Where is the American version of the aforementioned biotech startup?

This epidemic and the general response has only reinforced my belief in the future of citizen science and the widespread accessibility of indie biohacking tools and methodologies. There are just too many misaligned incentives in pharma research, leaving huge gaps in the ability to bring new tests and drugs to the market. Look no further than the antibiotic resistant drug problem.

[0]https://www.msn.com/en-us/finance/companies/a-berlin-biotech...

As far as I can tell, and admittedly this is conjecture based on piecing together a bunch of off-hand comments by official spokespersons and a few industry insiders I talk to regularly, it's because the U.S. decided that everyone needs to use the same exact test. There is some validity to that approach in that it makes the data a lot better for comparison tracking. But, they compounded that somewhat defensible decision to require that everyone uses the same exact test kit. This turned into a disaster when the chosen supplier couldn't produce the reagents in the required time/amount.

This an aside, but this seems like a textbook example of government regulation of healthcare not only failing but getting in the way and actively causing harm. There are literally 1000s of generally certified labs that they could have said very early on "go to it if you have this certification" and instead they plodded through this top-down heavy handed mess and made this pandemic many times worse.

In the US you have this combination of heavy over-regulation and lack of public provisioning. It's like the worst of both combined. Between this, cannabis, ketamine, and the opioid crisis, I absolutely do not understand why there isn't an uproar over overregulation in healthcare getting in the way.

None of those generally certified labs have any clue how to develop a new test. Not even a test that is exactly the same as a test they already have. There are 1000's of them, because you need very little training to push some buttons and enter data into forms. Those generally certified labs did what they are supposed to do. They looked to the government for guidance on what to test and how. The gov't made many mistakes: disbanding of the CDC pandemic response team. Putting anti-science Pence in charge. Forming a CDC team that was heavy on science and management but light on epidemiology. Not following the WHO's lead on their already established and working test. Listening to pharma's explanation about what they will be able to do in 12-18 months and confusing that with a statement about what they can do now. These are all mistakes that didn't have to happen and happened because morons are in charge.

As others have mentioned, false negatives and false positives are both annoying problems. The standard diagnostic kit for example comes with a positive control, which frankly is absolutely essential. Synthesizing a primer is trivial, a positive control is not as trivial (especially if it's an RNA sample). It's also an RT PCR, which means it's extremely easy to contaminate samples among each other (so you can never be sure if even one of your samples actually has the virus whether ALL of the samples have it or not). Further, you can't trust regular gel results too much, you need a qRT-PCR cycler which most labs would have but probably not these hackers.

Also were talking about a virulent pathogen sample. Do you truly want to stand in line and collect random snot samples with covid virus without proper training and PPE?

There's no stopping anyone from getting the primers (small pieces of DNA) and doing a qPCR yourself. You'll also need reverse transcriptase (the RT, converts RNA->DNA) and a way to purify RNA from a mix of cells/spit/junk. These are all relatively inexpensive, standard molecular biology reagents. There are some shortages of RNA extraction kits, but a good molecular biologist can do without one.

What you might be missing is consistency, which is what the medical system is worried about. You want to avoid false negatives AND false positives when it really counts. Getting that consistency is where the regulations come in. Nowadays I think it's appropriate to relax them a little in the interest of the emergency.

It's going to be hard to get the assay working and trusting without a positive control though

Correct! Though making that template isn't that difficult either, you can order the DNA version and generate the RNA from that.

> Given the simplicity of the testing process using PCR, why does it seem like the US is moving so slowly with getting tests in peoples hands?

This has already happened in the US, with 1.5 million tests ready to ship, with emergency approval from the US government. In April they will be producing 5 million tests per week. https://ir.thermofisher.com/investors/news-and-events/news-r...

Ready to ship and April are much different than the countries who have been, and continue to test, upwards of 10Ks+ folks a day. As of today, the CDC is reporting only N=4225 completed tests in CDC labs and N=20907 in other US labs.

General description of Mologic/Sherlock Bioscience technology:

"Mologic said it will invest a part of an expansion to its $4.9m grant from the Bill and Melinda-Gates Foundation to finance access to Sherlock's INSPECTR™ platform, which the Cambridge-based US company licences earlier this year from the Wyss Institute.

Under the agreement, Mologic will provide its lateral flow-based platform CARD, which has been demonstrated to provide a 1,000-fold improvement in sensitivity over current technology, and its immunoassay platform ELTABA for enzyme activity detection. Sherlock Bioscience will provide its synthetic biology-based INSPECTR™ platform, which consists of a DNA hybridization-based sensor that can be easily programmed to detect target nucleic acids (DNA or RNA) with single base pair specificity, coupled with a paper-based synthetic gene network that translates the sensor’s detection into a bioluminescent signal that is easily visualised or captured on instant film. Crucially, this process can be done at room temperature and does not require any instrumentation."

https://european-biotechnology.com/up-to-date/latest-news/ne...

"What is SHERLOCK™?

SHERLOCK is an evolution of CRISPR technology, which others use to make precise edits in genetic code. SHERLOCK can detect the unique genetic fingerprints of virtually any DNA or RNA sequence in any organism or pathogen. Developed by our founders and licensed exclusively from the Broad Institute, SHERLOCK is a method for single molecule detection of nucleic acid targets and stands for Specific High Sensitivity Enzymatic Reporter unLOCKing. It works by amplifying genetic sequences and programming a CRISPR molecule to detect the presence of a specific genetic signature in a sample, which can also be quantified. When it finds those signatures, the CRISPR enzyme is activated and releases a robust signal. This signal can be adapted to work on a simple paper strip test, in laboratory equipment, or to provide an electrochemical readout that can be read with a mobile phone."

https://sherlock.bio/technology/

Video of paper-based bioluminescence detection (Wyss Institute)

"If the sample contains the RNA of the Zika virus, the test area turns purple."

https://www.eurekalert.org/pub_releases/2016-05/uot-rld05061...

An alternative: "Anitoa systems, a Silicon Valley biotech company has demonstrated a solution for rapid on-site nucleic acid test of the 2019 novel coronavirus (2019-nCoV). Anita combines a high performance ultra-portable Real Time Polymerase Chain Reaction (qPCR) instrument called Maverick, with an innovative “one-step” reverse transcription qPCR (RT-qPCR) reagent for testing 2019-nCoV."

http://www.anitoa.com/docs/press-ncov.pdf