7 ms·

From their website [0] my guess is their test is ELISA [1] based or similar method of detecting if the human body has had an immune reaction to the virus in the

From their website [0] my guess is their test is ELISA [1] based or similar method of detecting if the human body has had an immune reaction to the virus in the amount that is detectable. Where as the more expensive test is PCR based [2] that requires the heating-cooling cycles and machines to do it. The PCR-based test basically take the RNA of the virus converts it to DNA and then doubles the amount of DNA every heating cycle. Assuming we are talking about real-time PCR the PCR cycle of double is what takes time. I have not seen the data on the incubation period and the immune response, to know if these immune detection tests will be able to provide early detection for asymptomatic case . It's probably for symptomatic cases and wanted to ensure it's not flu or another infection and it's COVID-19. This is not to say the test is useless. It actually great inexpensive way of testing the population with symptoms and using more expensive and timely test for early detection. Reducing the overall burden. worth mentioning are other initiatives that are focusing on cheaper and faster PCR process (though with potential of higher false negative and positive results) [3] [0]https://mologic.co.uk https://mologic.co.uk [1]https://en.wikipedia.org/wiki/ELISA https://en.wikipedia.org/wiki/ELISA [2] https://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction https://en.wikipedia.org/wiki/Real-time_polymerase_chain_rea... [3] https://twitter.com/JMRothberg/status/1238115465467133959 https://twitter.com/JMRothberg/status/1238115465467133959

entee 7y ago
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
- maxerickson 7y ago
  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)
  - entee 7y ago
    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-test https://www.propublica.org/article/cdc-coronavirus-covid-19-...
- gewa 7y ago
  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.
joshuamcginnis 7y ago
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-company-got-a-head-start-on-coronavirus-tests/ar-BB114rEO https://www.msn.com/en-us/finance/companies/a-berlin-biotech...
- hcknwscommenter 7y ago
  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.
pdm55 7y ago
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/news/sherlock-bioscience-and-mologic-enter-partnership.html https://european-biotechnology.com/up-to-date/latest-news/ne...
- pdm55 7y ago
  "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/ https://sherlock.bio/technology/
  - pdm55 7y ago
    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-rld050616.php https://www.eurekalert.org/pub_releases/2016-05/uot-rld05061...

entee 7y ago

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

maxerickson 7y ago
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)
- entee 7y ago
  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-test https://www.propublica.org/article/cdc-coronavirus-covid-19-...
gewa 7y ago
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.

joshuamcginnis 7y ago

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-company-got-a-head-start-on-coronavirus-tests/ar-BB114rEO https://www.msn.com/en-us/finance/companies/a-berlin-biotech...

hcknwscommenter 7y ago
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.

pdm55 7y ago

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/news/sherlock-bioscience-and-mologic-enter-partnership.html https://european-biotechnology.com/up-to-date/latest-news/ne...

pdm55 7y ago
"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/ https://sherlock.bio/technology/
- pdm55 7y ago
  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-rld050616.php https://www.eurekalert.org/pub_releases/2016-05/uot-rld05061...