4 ms·

Ask HN: Any biomolecular/bio-lab hackers working on DIY RT-PCR tests?

4 points by yourapostasy 7y ago

From a layperson's perspective, it looks like purchasing Covid-19 primers, setting up my own thermocycler and gel electrophoresis chamber would let me test my community myself. If I could run 16 tests per person (8 nasal, 8 pharyngeal) to manage false negatives in this time of high viral activity, as long as the equipment was calibrated and validated, then I'd rather do that than wait for a bumbling administration to trickle down tests to my locality (despite the flowery words in the Rose Garden just now), if they even show up in time at all to flatten the R0 curve. But I'm just a layperson. Where are bimolecular hackers gathering to talk about the concrete steps it takes to not only set this up, but calibrate it (for example, where do I source the calibration dyes and buffers I need to use), and validate its accuracy and precision, etc.? I've found there are a number of primer suppliers, but I'm also interested in whether or not hackers are assembling their own primers, or even those looking into CRISPR-based detection [1]. [1] https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf

rolph 7y ago
heres some reading to get started. you dont need the electrophoresis unless you want to do something like restriction analysis across the different samples. mainly its a test for presence of sars-cov2. very often this involves annealing a molecular reporter such as a dye florescence probe, to the mRNA with specificity to regions characteristic of the strain your looking for. https://www.researchgate.net/profile/Idania_Gonzalez-Perez/publication/6684862_Homemade_viral_RNA_isolation_protocol_using_silica_columns_A_comparison_of_four_protocols/links/5a82eeeda6fdcc6f3eae496c/Homemade-viral-RNA-isolation-protocol-using-silica-columns-A-comparison-of-four-protocols.pdf https://www.researchgate.net/profile/Idania_Gonzalez-Perez/p... https://www.fda.gov/media/134922/download https://www.fda.gov/media/134922/download --------------------------------------------- https://hackaday.com/?s=PCR https://hackaday.com/?s=PCR https://hackaday.com/2016/05/03/dna-extraction-with-a-3d-printed-centrifuge/ https://hackaday.com/2016/05/03/dna-extraction-with-a-3d-pri... https://hackaday.com/2013/09/23/openfuge-an-open-source-centrifuge/ https://hackaday.com/2013/09/23/openfuge-an-open-source-cent... https://hackaday.com/2013/09/21/vcr-centrifuge/ https://hackaday.com/2013/09/21/vcr-centrifuge/ https://hackaday.com/2020/03/10/open-source-collaboration-tackles-covid-19-testing/ https://hackaday.com/2020/03/10/open-source-collaboration-ta... ------------------------------------------- https://news.ycombinator.com/item?id=22552391 https://news.ycombinator.com/item?id=22552391
- yourapostasy 7y ago
  Thank you! Your last link is exactly what I was seeking, greatly appreciate it. See omar_a1's feedback, sounds like he's pretty certain it's impossible. Meanwhile, I'm wondering what Bento Lab or other synbio kits can do for self-testing if a complete from-scratch DIY option is time-constraint infeasible. Thanks again.
  - rolph 7y ago
    bento lab is an interesting idea for a field test in remote regions, especialy for surveillance work. it would have to be acceptable to be expendable, and it is better in the long term to have these instruments in separate form. https://en.wikipedia.org/wiki/Thermal_cycler https://en.wikipedia.org/wiki/Thermal_cycler https://en.wikipedia.org/wiki/Laboratory_centrifuge https://en.wikipedia.org/wiki/Laboratory_centrifuge https://en.wikipedia.org/wiki/Gel_electrophoresis https://en.wikipedia.org/wiki/Gel_electrophoresis the big issue would be continuity of reagents, and perhaps forming some just in time method for preparing wet solutions from dry stock.
saluki 7y ago
Other than for statistics and tracking the spread are tests really needed at this point? Is there an advantage to seeing a positive/negative test result for a patient. As there isn't a treatment plan and it seems like recommendations are for anyone with cold symptoms to self quarantine for 2 weeks+. I feel like it's so wide spread now we just have to ride it out, use social distancing, avoiding events/meetings to try to minimize/stretch out the peak number of cases. Maybe save the test kits for when we do have a reliable treatment. Or better yet if the treatment is safe and inexpensive hand it out like candy.
- yourapostasy 7y ago
  I'm wondering if decentralized DIY testing can arise from centralized, vetted plans giving skilled tradespeople enough information to construct, calibrate and validate their own tests. And if enough can be made to reasonably rule out false negatives before positives are sent onwards to "official" testing, and lessening the inevitable load on hospitals, and in these early days of testing, the testing facilities. Everything I've read so far doesn't seem to say "first, you need a <fission-reactor-scale-dooodad>". So I'm wondering how feasible it is, and the deeper I dig into this, the more I'm wondering just how low the bar goes.
omar_a1 7y ago
Uh, sorry to be that guy, but if you have no experience with lab techniques, microbiology, lab instrumentation, or analytical method validation, this isn't the sort of thing you can reasonably expect to accomplish without dedicating several years of your life to getting the necessary technical skills, to say nothing of the huge sums of money it would take to set up your own lab. It's kind of like deciding you want to build your own 3 story house by hand. You can do it, but buying a bunch of lumber and tools is a far cry from having the necessary skill to actually build it. Science is hard. There's also a major ethical issue about the medical implications of testing someone for a stigmatized disease if you're not clear on the validity of the test. False positives and false negatives have a major impact on your community. And hand-waving away the implications of reporting a false positive or negative with the statistical caveats of the results doesn't cut it. Patients tend to take test results at face value, so the responsibility to of weighing the ethical ramifications of when it's strictly necessary and when it's irresponsible to administer an unreliable test for a given patient fall squarely on your shoulders. (Not that that excuses the current administration's policy of negatively biasing reporting by not testing anyone.)
- yourapostasy 7y ago
  Appreciate the input. Ethical issues: I'm thinking in terms of underserved communities where tests are simply unavailable. A 60% false negative rate test is better than no information whatsoever. After all, it was good enough for China to use (along with authoritarian powers to quarantine) to help get a handle on containment, or what am I missing? Considering the abysmal US track record serving the healthcare needs of lower socioeconomic strata, I'm not sanguine on them receiving the tests we need to even get an approximate handle on the public health situation. So if we narrow the scope to just testing for Covid-19, are you saying it still takes several years to get to the point of just administering and evaluating the test, right? If so, then that means testing is staffing constrained, and you're implying it is impossible in a situation like we face now to dramatically expand the testing scale/volume, and the entire US Federal plan to test orders of magnitude more is complete bullshit. So is this one of the few areas of human endeavor that is absolutely non-automatable, or can we break down what is/is not within the realm of automation, and possibly leverage the few staff we have relative to the current need? Or can we relax quality constraints in an emergency like this to favor false positives over false negatives to focus on flattening R0 (although everything I've read so far seems to indicate false negatives are the bigger concern)? So please give some pointers, where do you see specific, concrete problems for starters that others with domain expertise can look into to evaluate?
MoodyTU 7y ago
Just some thoughts Even if you develop a perfect test, it's unlikely to help the people you're testing. People who are feeling sick (and those who aren't) should be staying home and minimizing contact with others, and going to a hospital if they are sick enough to require doing so. A positive or negative test data point doesn't change that. The data could be useful epidemiologically, but only if you can convince others to use your data. That will likely be difficult to do. Most importantly, you're very likely to get sick yourself handling 10s or 100s of samples without training and appropriate facilities. Totally appreciate your drive, but your time might be better spent contacting your representatives and making it clear we expect better from them.

omar_a1 7y ago
A 60% false negative rate test is better than no information whatsoever. No! It's decidedly not better! This isn't web-scraping where any little bit of information you extract is good, even with a low signal-to-noise ratio. These are human beings. Being wrong here has serious consequences, to the point that you need to weigh the usefulness of the test against the risk of harm of misdiagnosis. You can't just go around diagnosing people willy-nilly if you're not sure about the results! A false negative means someone will go out with a false sense of security, potentially interacting with vulnerable community members thinking they'll be fine. They may develop further complications due to writing off their symptoms as the flu, and not seek out medical intervention if the situation goes South. A false positive means someone is going into quarantine for two weeks at a major economic and housing consequences (particularly if you want to help poor communities). False positives and false negatives mean more lives will be lost. The stakes are higher, and so too is the need for ethical rigor. So if we narrow the scope to just testing for Covid-19, are you saying it still takes several years to get to the point of just administering and evaluating the test, right? I'm not saying the test will take several years to develop, I'm saying it would take you, a self-described layperson, several years to gain the technical skill and know-how to know how to correctly research, design and validate the test. Specifically, labs are carefully controlled environments. Just getting into the grunt work mechanics of the technique, at bare minimum you need to learn aseptic technique, pipetting, sterilization, reagent prep, handling hazardous chemicals, biohazardous waste disposal, and establishing appropriate environmental controls for your lab. Starting from nothing, those will take at least a year of full-time experience to develop for any semblance of competence. Otherwise you'll be a bull in a china shop; your results will be too tainted by bad technique and inadequate environmental controls to get anywhere. So is this one of the few areas of human endeavor that is absolutely non-automatable, or can we break down what is/is not within the realm of automation, and possibly leverage the few staff we have relative to the current need? It's perfectly automatable, but that doesn't mean it's trivial. For one, someone needs to... actually automate it. Someone else needs to implement, maintain and troubleshoot the robotic lab equipment doing the automation. Still require highly specialized skills to do so.