Contents

1 Establishing CDC protocol at Gloriastrasse

Gloriastrasse has the Lightcycler 96, which is already accredited for diagnostic qPCR and the Lightcycler 480 which is used for research. Both have channels for FAM and VIC and 2 others.

We tested Antonia’s sample and the positive control in both machines.

Both Lightcycler 480 and 96 have very similar CT values as compared to the Viia7 in Schlieren.

Aizhan had to dilute the input RNA 1:5 to preserve the samples, so the decrease in about 2 cycles is expected.

## Using 'Ct.Mean' as value column. Use 'value.var' to override

2 Testing real samples with the Lightcycler 96

We received 30 samples from Philipp to test. These were already tested by Virology.

The Qiacube was used to extract the RNA.

All samples have Rp detection so the extraction was successful, and we could detect all 3 N genes in each of the positive sample at varying CT values.

## Using 'Cq' as value column. Use 'value.var' to override
## Using 'Cq' as value column. Use 'value.var' to override

Since we had the Virology results we could directly compare. Virology has a threshold for weak and strong positivity. They said weak was anywhere from 1-200 fold of the detection limit and strong was more than 200. In our case, if we assume 40 cycles as our limit, then 8 cycles is about 256 times higher so we will set 32 cycles as the cutoff between weak and strong.

We had 100% concordance with Virology. There was only one sample where we classified the sample as strong positivty compared to weak in Virology. This sample had CT values of about 30, so close to the threshold.

2.1 Testing with Kingfisher extraction

The remaining aliquot from the virology samples were extracted with the Kingfisher. Not so much material was left so there were varying levels of input. From < 50 uL, about 50 uL, and 50-100 uL. Most of the CT values were comparible, except for sample 956 which had about 3-4 cycles of difference and the sample had low N genes, so N1 wasn’t detected anymore.

Even with the lower amounts of input, our cutoffs are still in the “high” range for all the high samples. Sample 956 was labelled “Retest” because N1 wasn’t detected. There were 5 samples that weren’t available for the Kingfisher extraction.