Contents

1 COVID19 PCR testing

1.1 Mouse primer test

To have a internal PCR control, we would spike in some foreign DNA to the test sample and also include a probe/primer set in a different color in the well.

The CDC doesn’t provide this internal PCR control, so we have to design it ourselves. Aizhan has some DNA and primers for mouse INS6 and GAPDH. She tested the CDC primers N1, N2, N3 and RP against the mouse DNA to check for specificity as well. Surprisingly, RP was also amplified in the mouse DNA.

1.2 CDC test

Next we performed a titration curve to check the efficiency of the PCR for the coronavirus primers. The CDC has 3 primers targeting the N (nucleocapsid) gene of the SARS-CoV-2 virus.

The efficiency looks good. The intercept is almost 40 which means the primers could theoretically detect 1 copy of the N gene.

Unfortunately, the GAPDH for mouse is detected in the human control DNA. However INS6 is still specific for mouse DNA.

1.2.1 Tests with positive Corona cases

We tested positive samples from Antonia and negative samples from Muriel and Philipp. Using different extraction methods. Kingfisher with GE beads, manual Qiagen column, Maxwell 16 DNA blood, and Trizol. Trizol had the worst performance, in general it had 5-7 higher CT than the rest of the extractions and Muriel’s Trizol was positive for the corona genes.

The automated extraction methods were very similar to each other, so we can use either one.

We also wanted to see if there was correlation between RNA content and CT value, however there doesn’t seem to be.

1.2.2 Spike in with mouse RNA

Since the CDC assay currently doesn’t have a internal PCR control in the same well as the probe, it only has RP in a separate well for the PCR control, we spiked in mouse DNA and use mouse GAPDH to determine if we can use it.

Unfortunately, like the previous GAPDH experiment, Human DNA has a positive signal with mouse GAPDH, although at much higher CT values.

We will have to design a proper internal PCR control.

1.2.3 Test with other positive samples

New beads manufactored from ETH were tested for their efficiency in RNA extraction with the Kingfisher robot.

We also had 5 new positive samples from Philipp, which were all positive using the GE Bead extraction method.

All custom beads from ETH were slightly less efficient than the GE Beads, about 5 cycles more.

We had a small contamination of the water well which was then positive for RP. Luckily, DEPC was negative for all genes.

Philipp’s negative control sample also worked for all extraction methods, although B Bead did have a positive signal for N2, this is probably just some pipetting error, as all other extractions were negative for all N genes.

1.2.4 Testing again with mouse spike in

We tested the CDC protocol again with Rim3 mouse DNA spike-in using mouse GAPDH in the VIC channel. All N genes are in FAM channel.

We used Antonia 3 and a the positive control for CDC. All Coronavirus genes were detected except for a possible pipetting mistake in Postive control for N2.

Mouse GAPDH was detected in all samples.

1.2.5 Qiacube vs Kingfisher

Philipp acquired a Qiacube to process the samples automatically through the Qiagen columns.

He extracted the previous 5 sample hes provided to Ossia for the Kingfisher extraction.

Philipp had two replicate samples from the Qiacube extraction and 1 sample from the GE Beads

Both extraction processes are very similar. Although for Pat2, which has CT values about 39-40 in the Kingfisher extraction, the Qiacube failed to have any detection.

1.2.6 Scale down and using Invitrogen polymerase

Scale down works. We get the same CT values from the 10 uL reaction volumes as in the 20 uL reaction volumes. We also tried using the Invitrogen Taq polymerase mix, using the Pasteur protocol, but it did not work, it also amplified all genes in the negative water control.

Retesting Pat2 again, we can detect N1 and N3 transcripts but not N2.

1.3 Bead testing

ETH has some beads to test for DNA/RNA binding. We used Antonia 1 and Muriel as a positive and negative control respectively to test a variety of beads.

1.3.1 combine with previous bead data

I could combine this run with the previous bead run data for Antonia’s sample. Unforunately, Muriel wasn’t the negative control from the previous run so we can’t compare. It seems this batch of beads isn’t as good as the B bead for viral gene capture. At least the cycles between the RP and N genes are closer with this batch of beads.

1.4 Testing Pasteur protocol

The Pasteur protocol has two probes for COVID19, IP2 for the RdRP (RNA dependent RNA Polymerase) gene and the E (Envelope) gene. The IP2 probe is in the VIC channel and E is in the FAM channel. In this experiment we also have RnaseP in FAM and VIC channels as the interal PCR control. We can combine RP VIC with E as a way to reliably have a negative result for E and can combine RP FAM with IP2 as a way to reliably have a negative result for IP2.

The samples we used were the positive control from the CDC, the positive control for the multiplex thermofisher, and RIM3 dna for mouse.

The RP FAM did not work in the positive control for CDC, but the datasheet from CDC states RP doesn’t necessarily need to be positive in their control DNA.

RP VIC worked in combination with E for the thermofisher control but at both 25000 copies and 50 copies at similar CT values which makes us suspicious of the result. RIM3, human and the negative control also had a positive hit for RP VIC + E. Which really suggests that this primer/probe set isn’t specific.

1.5 Thermo singleplex test without Thermo positive control

We also tested the thermo singleplex probes but without a proper positive control. Thermo singleplex has probes for orf1ab, N, and S (Spike glycoprotein) regions of the coronavirus.

We tried to use the multiplex control from thermo as a positive control, however after consulting with thermo, they said the positive controls from each kit were unique. Nonetheless, we did detect positive signal at two different levels 25000 copies and 50 copies in RP and only positive signal in N at 25000 copies.

Rim3 also had positive hit in Rp. Surprisingly, human and mouse gapdh work in both Rim3 and human DNA.

We also had some positivity in RP in our negative control.