TITLE: Corona Detective User Protocol V2.0
AUTHORS: Rachel Aronoff, Guy Aidelberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Corona Detective</span><span> is based upon a molecular amplification strategy inspired by the ‘</span><a href="https://gmodetective.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">GMO Detective</span></a><span>’ method, in order to detect the virus causing Covid-19. Done not only without complicated equipment, but with a simple +/- fluorescent readout, the </span><span style = "font-weight:bold;">Corona Detective</span><span> is very specific. Furthermore, controls to ensure sensitive detection, without false positives or negatives, are intrinsic to this solution. The final product, strips/plates of tubes with dry reagents, specific for Corona and a control gene (extraction control), can be shipped anywhere, without cold-chain dependence. Critically, monitoring tests could be run by ordinary people, by following this protocol. (Even kids have successfully participated in GMO detective workshops, also soldering their own fluor detectors.)</span></div><div class = "text-block"><span>This user guide will allow you to test samples with this system, provided as freeze-dried reaction components in 0.2ml tubes made by this </span><a href="https://www.protocols.io/view/freeze-drying-lyophilization-and-manufacturing-of-bpv4mn8w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocol</span></a><span> (other formats possible  e.g. 8-tube strips, 96-well plates, 1000 reactions, etc.).</span></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><span style = "font-weight:bold;">Human or clinical samples should only be run in settings with access to appropriate biosafety facilities, of course.</span></div><div class = "text-block">Acknowledging the JOGL Open Covid 19 Initiative and all the #proj nucleic-acid-amplification team for support.</div></div>

[STEPS]
?. Sample Extraction: Two options are possible A:  Direct reactions of inactivated sample (i.e. i.e.  saliva, with the addition of 1/100 100x TCEP/EDTA inactivation reagent, and after incubation at  for ) as in Rabe and CepkoB:  RNA Concentration with 'Magnetic Beads' or other validated extraction methods as described or here.The B method for Sample Extraction may be especially for highly sensitive detection of SARS CoV-2 virus (N gene target), but the A method is simpler, and very sensitive, (and may be useful also for certain scenarios, like surveillance screening: especially if one would prefer to identify only people who might most be expected to currently transmit virus).  Extraction/concentration methods (B) take longer and are more expensive in addition to requiring a bit more infrastructure and careful elution to add the sample to Corona Detective components but can improve sensitivity (especially if pooling samples for testing).100x TCEP/EDTA (from  Rabe and Cepko)To make 5 mL of 100× inactivation reagent, first,  of TCEP-HCl (Millipore Sigma 580567) was dissolved in water to create  of a  solution. Then  of 0.5 M EDTA, pH = 8 (ThermoFisher Scientific AM9260G) was added. Finally, of 10N NaOH is added and UltraPure water to bring the final volume to.- of inactivated A or eluted B sample is used in each   CoronaDetective reaction
100 µl
95 °C
358 mg
2.5 mL
1 mL
575 µl
5 mL
4 µl
20 µl
?. Isothermal amplification  Set up the water bath or other incubators, in order to incubate reactions at and at Celsius.  Many options for achieving stable incubation temperatures exist. Anything from a thermocycler, to precise heating blocks or even other options, like a sous-vide precision cooker.Set up the reactions.Label carefully, including at least one positive and negative control. (Corona Detective 8-tube strips can be produced so that they include these control samples already.)Make sure lyophilized pellets in the tubes are at the bottom of all the tubes before carefully opening them. Open the tube containing rehydration buffer and add  (provided with Corona Detective) into each of the reaction tubes. (For the 96 well format, a multipipettor or dispensing pipette is useful for this step).  Add  of clean water to designated negative control tube/s (closing lids as you go when possible).  Add  of clean water to designated Positive control tube/s, with included freeze-dried DNA control (or add  your own control solution, for instance, RNA controls, which are not expected to survive the lyophilization. )Then, add of the test samples (from step 1)  to the rest of the test tubes.Mix Reactions Well. You can even vortex gently and either spin down or flick down, tapping gently.  Incubate the Corona Detective tubes for  at , to allow RTx to reverse transcribe the sample RNA to DNA.  Incubate the Corona Detective tubes at for at least   (to  ) and cool to   for Detection (step 3).  If not reading results right away, tubes can be placed in a fridge  . The results are stable for weeks even at  .
55 °C
64 °C
[Rehydration buffer]
4 µl
4 µl
4 µl
4 µl
MAKE SURE ALL WELLS ARE  SEALED WITH APPROPRIATE CAPS OR ADHESIVE SEALING FILM!
55 °C
64 °C
0 Room temperature
4 °C
0 Room temperature
?. Detection:  Cool tubes to   or less, then place the tubes in a fluorescence detector (e.g. DIY GMO Detective Detector, gel transilluminator, or other) and take a picture.  Tubes positive for the presence of SARS CoV-2 N gene will exhibit bright green fluorescence. The negative control (no template) should remain 'dark,' while designated positive viral control tube should exhibit bright green fluorescence.  A weaker orange fluorescence will confirm the presence of human cell RNA, especially if there was no virus RNA detected. This controls for extraction efficiency and should not be seen in negative control tubes.  QUASR fluorescent signal develops as the short quencher primers are displaced through amplification of product, with the N-target labeled with the FAM fluorophore, a blue LED excitation filter and amber emission filter are ideal (as in the GMO detective system).  The internal control for RNAseP is labeled with the HEX fluorophore, which can also be seen (more orange, less green) with the DIY Detector from GMO detective.Single primer set reactions and then the multiplex reactions are shown below as examples:Removing the blue filter and including a more orange emission filter, allows these two signals to be distinguished, even on a cell phone camera (an old iPhone SE was used above).  However, red (tagging alternatively the RNAseP primer with a TexasRed fluorophore) may also be used to distinguish the two signals better, with the same blue (or green) excitation and a red emission filter.Avoiding saturating the camera sensor is important, to make sure you can distinguish negative from positive results. (Auto-adjusting exposure, if not automatic, can usually be initiated with a tap on a bright point of the image.)Old sample tubes from this reaction, if protected from light and simply kept at room temperature, can be looked at weeks later with no obvious change in the resulting fluorescence. If the positive control tube from the experiment does not exhibit bright green fluorescence, or the negative control does, the experiment has failed and needs to be repeated.
0 Room temperature
Never open the used tubes after reactions have been run!  To dispose of used tubes directly after pictures are saved is recommended.