TITLE: Viral RNA extraction low-cost protocol optimized for SARS-Cov2 at AGROSAVIA
AUTHORS: Alejandro Caro-Quintero,   Roxana Yockteng 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we present the second version of an RNA extraction protocol for SARs-cov2 using magnetic beads. This protocol was optimized in AGROSAVIA laboratories and is presented as an alternative to the limitations of commercial viral RNA extraction kits. The viral RNA extraction protocol was adapted from the protocol entitled “SARS-CoV-2 RNA purification from nasal / throat swabs collected in Viral Transfer Media” at https://bomb.bio/ and which is based on methods previously reported by the same group (Oberacker et al 2019). The optimized version here achieves Ct results like the commercial kits and reduces the cost of RNA extraction per sample by 90%, that is, 2000 Colombian pesos or 50 cents per sample. We share this protocol in the hope that it can ensure the sustainability of the diagnosis in Colombia and other countries where it is difficult and expensive to import commercial kits.</div><div class = "text-block"><span style = "font-weight:bold;">REFERENCES</span></div><div class = "text-block">Oberacker, P., Stepper, P., Bond, D. M., Höhn, S., Focken, J., Meyer, V., ... & Hore, S. R. (2019). Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLoS biology, 17(1), e3000107.</div><div class = "text-block">Yu, F., Qiu, T., Zeng, Y., Wang, Y., Zheng, S., Chen, X., & Chen, Y. (2018). Comparative evaluation of three Preprocessing Methods for extraction and detection of Influenza A Virus Nucleic Acids from sputum. Frontiers in Medicine, 5, 56.</div><div class = "text-block">He, H., Li, R., Chen, Y., Pan, P., Tong, W., Dong, X., ... & Yu, D. (2017). Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections. Scientific reports, 7, 45199.</div><div class = "text-block">Corman, V.M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D.K., Bleicker, T.,Brünink, S., Schneider, J., Schmidt, M.L. and Mulders, D.G., 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance, 25(3).</div><div class = "text-block">Kumar, M., Mazur, S., Ork, B.L., Postnikova, E., Hensley, L.E., Jahrling, P.B., Johnson, R.and Holbrook, M.R., 2015. Inactivation and safety testing of Middle East respiratory syndrome coronavirus. Journal of virological methods, 223, pp.13-18</div></div>

[STEPS]
?. Pre-processing of mucous samples.
?. Dilute of the sample in of Saline Solution [, vortex mix  . Use only of this dilution to star protocol. In step 2.3 add  isopropanol after incubation.
100 µl
300 µl
100 µl
540 µl
?. Sample Processing (in 1.5 ml tubes)
?. To  of GITC buffer add  of Proteinase K ], of the sample and mix by vortex for .
100 µl
12.5 µl
200 µl
?. Incubate the sample for  at , vortexing every .
60 °C
?. Place the tube in a rack and add  of Isopropanol and  of magnetic beads (see preparation below). Mix by inverting 8 times.
270 µl
40 µl
?. Place the tube (s) in the magnetic rack for without moving.
?. Without moving the tube from the magnetic rack, withdraw approximately  of the solution
610 µl
?. Without moving the tube from the magnetic rack, add of Isopropanol and wait 30 seconds and remove the isopropanol from the tube.
150 µl
?. Without moving the tube from the magnetic rack, add of Ethanol, and remove from the tube.
200 µl
?. Without moving the tube from the magnetic rack, repeat previous washing 2.7 and extract the Ethanol to the maximum.
?. Without moving the tube from the magnetic rack, allow drying for approximately .
?. Add l of molecular grade water to the tube trying to discharge the liquid on top of the tube walls where the beads are located.
30 µl
?. Remove the tube from the magnetic rack and vortex  to allow the beads to enter the solution and incubate  at room temperature in a normal rack
?. Place the tube back in the magnetic rack and for  to , and transfer of the bead-free elution to a new tube and store at.
20 µl
-80 °C