[Peter Stoilov]

Hi all,

 

first of all thanks to the BOMB team for the excellent protocol!

We tested a slightly modified protocol for RNA extraction efficiency and sensitivity using in vitro transcribed RNA for the SARS-nCov2 virus N-gene (CDC primer sets).

The modifications are as followed:

1. GITC solution is diluted by 10% to prevent precipitation. The volume is adjusted accordingly to 110ul lysis buffer per 200ul of sample.

2. The magnetic beads are diluted 25 fold, and their volume is reduced by two fold. This compensates for the increase of the GITC solution volume, so that the volumes for rest of the reagents do not need to be changed.

3. Incubation time on the magnet is longer: up to 25-30minutes with mixing at 10min. This depends on the strength of the magnet, so you have to pay attention and make sure you pulled down all of the beads before you start washing. I suspect this is the reason why some see lower recovery than column kits.

4. We use water instead of TE to elute the nucleic acids. We attempted to reduce the elution volume, but at least on our plates it was hard to recover the liquid when the volume was too low so we stick with 50ul elution volume.

5. We multiplexed the CDC primer set and put all three primer sets together with FAM, VIC and ABY labeled TaqMan probes.

6. We use Thermo TaqPath single tube mastermix (4x) and put 10ul of the eluted RNA per reaction.

 

With the protocol modified as described we see near complete recovery of the RNA. The limit of detection is 145 copies per ml (call it 200 if you want to be conservative). We call a positive hit if at least one out of the three probes gives us a positive signal. At 145 copies per ml 90% of the reaction are positive for at least 2 probes and 100% are positive for at least one probe. At 15 copies per ml 50% of the reactions are positive for at least one probe. To put this in perspective, at 15 copies/ml starting material before the purification and assuming 100% recovery, a PCR reaction would contain 0.6 copies of RNA meaning it has 60% chance to have one RNA molecule. The calculation is as follows: 200ul x 15 copies/ml= 3 copies; 10ul out of  50ul containing 3 copies = 0.6 copies per reaction.

We used a simulated virus, lentiviral particles with cloned SARS-nCov2-N gene in VTM, and the viral RNA also seems to be recovered efficiently. I will have hard data on the LOD for the viral particles in the near future.

The sensitivity can be increased by using larger volumes. Deep well plates with capacity of 2ml will in theory allow using 400-500ul of sample and double the sensitivity. The downside is that this  setup is harder to handle: deep well plates require longer tips, make it harder to reach the bottom, don’t sit well on the magnets, and the volumes exceed the capacity of the multichannel pipettes thus requiring more pipetting.

Finally, we did tests on the Qiagen viral RNA extraction kits and the recovery there is 67% of the input. So, in our hands the BOMB-COVID19 protocol is performing better that the Qiagen kit with the added benefit of not needing carrier RNA (does anyone know the origin of the carrier RNA in the Qiagen kits?).

 

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