@kyliemdrake
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Thanks Tim.
Alexander – Tim M is correct. Our focus is on delivering a diagnostic service at present. However, I will take your comments under consideration and we’ll see what our team can do.
To answer a couple of questions though:
Our validation was compared to the Abbott RealTi<i>m</i>e SARS-CoV-2 kit and extraction on an Abbott M2000. The Ct values for positive controls differ by 1-3 cycles (with the BOMB data coming up later) – this is likely due to a decrease in the amount of input VTM/UTM though, not a decrease in extraction efficiency.
We are using GE Healthcare Sera-Mag Magnetic Speedbeads (GEHE4515210).
We have not yet moved this method to any of the liquid handling systems that are currently available to us due to the logistics of sourcing appropriate plasticware. When we do, I will make sure we make our protocols available to you.
Cheers,
Kylie
Emily,
Ideally, collecting straight into GITC will solve the volume problem. But we had to make do with current practice in our laboratory. Who is adding your swabs to VTM? If it isn’t happening at the clinic, consider a move to GITC.
One way to reduce your input volume is to increase the molarity of the GITC lysis buffer. That’s why the COVID-19 protocol uploaded here uses 6M GITC. If you maintain a 2:1 ration of VTM:GITC and a 5:4 ratio of beads+GITC+VTM:isopropanol you should be able to reduce your input volume. just be careful that the 6M GITC doesn’t precipitate.
We started off in a 2.2mL deep well plate and the volume was too high to facilitate settling of the beads. The ability of the magnet to pull down the beads decreases exponentially the further away from the magnet you are. So unless you have an automation system with a magnet that surrounds your entire well (Abbott m2000), or has a magnet that can be immersed in the well (Kingfisher), you’ll have trouble getting the beads to pellet with such a large volume.
One trick that worked is to “clarify” the solution by slowly pipetting the volume past the magnet. Bringing the beads closer to the magnet will allow them to be “captured”. This works to an extent, but we still found that it took upwards of 45 minutes to pellet the beads in a large volume (>1mL). Which is ultimately why we settled on a 0.8mL plate and a smaller volume. You can then use either a ring magnet or something like this the Invitrogen™ AM10027 magnet. The AM10027 is what we’re using at Canterbury Health Labs.
To date we haven’t encountered any issues with clumping of the beads. Although we’ve yet to try extracting from sputum samples. I wouldn’t think there were enough cells in a throat or nasopharyngeal swab to cause clumping.
Good luck!
Kylie
