Data and results

Sars-Cov-2 sensitivity test

Start material, RNA extract from Trondheim. First, a large batch (1000µl) of cDNA was created and used as template in all the reaction (to omit variation due to cDNA synthesis). Thus, the tests are designed to test the efficiency of the primer stes. The cDNA mix was made of the following components; 2µl RNA, 200µl qScript mix (5x) (contains buffer and dNTP), 1µl reverse primer E gene (#21), 1µl reverse primer N gene (# 7), 786µl H2O and 10µl MashUp (reverse transcriptase). The solution was incubated at 50°C for 60 minutes.

Three 96 well plates were used in the dilution series;

Plate # 1, 10µl cDNA plus 100µl qPCR MIX and serial diluted by a factor of 2

Plate # 2, 1µl cDNA plus 100µl qPCR MIX and serial diluted by a factor of 2

Plate # 3, 1µl cDNA plus 100µl qPCR MIX and serial diluted by a factor of 3

Recipe for the qPCR mix, each well revived a total of 10 µl mix

PCR temp cycling program: Initial boiling, 94°C-1min, 45 cycles of 94°C-20sec, annealing 56°C-20sec, extension 72°C-40sec (picture), after the final cycle melting analysis was performed.

Amplification curves for 12 different cDNA concentrations (click to enlarge).

Plate #1
Plate #2
Plate #3

Wells with higher cDNA contraction give signal first. Below is the comparison of mean Cq value for 3 replicates in relation to cDNA concentration for the two primer sets teste. At lower cDNA concentration, the Cq value increases. Due to stochastic variation in the PCR reaction, it is not possible to quantify the number of starting molecules using qPCR. The signal generated depends on the PCR efficiency and number of copies amplified in the first few cycles. The data show that the PCR efficiency changes at low concentrations of cDNA. The theoretical lower template amount (pr. µl) that gave a signal was 1/(9*10⁹) of the original RNA  in the Trondheim sample. This is under the assumption that all RNA copies were converted to amplifiable cDNA in reverse transcriptase synthesis.

Amplification curves for the 3 lowest cDNA concentrations with 3 replicates each for the two primer sets used.

N-gene, #7	E-gene, #21

Limiting dilution

Start material, 2µl RNA from the original Trondheim in a 1000µl cDNA synthesis reaction. 1µl of this solution is diluted in 200µl H₂0, 1µl of this dilution is diluted in 200µl H₂0. From the last dilution, 10µl was used as template in a 500µl qPCR mix. The dilution from the original RNA then becomes. 1/500 * 1/200 * 1/200 * 10/500 which becomes 1/(1*10⁹) per µl. Since there is 10µl qPCR mix per well, we start with 1/(1*10⁸) of the starting material. Simultaneously a 1/(5*10⁹) was made. Primer set #21 was used for the amplification.

Target amplification was determined by melting curve analysis. Correct amplified DNA will go from double strand DNA to single strand DNA at a very temperature specific manner. Thus, unspecific amplification product will melt at different temperatures. This will give a different signal in the DNA melting point analysis. Below is the melting point analysis for the fragment amplified with primer set #21. The example shows the derivative melting curves when enough templates have been added. The average melting temperature is 80.8°C (STD 1.3). In the analysis with low template samples, peaks with a melting temperature between 81°C to 82°C were considered to be "true" amplification product.

Dilution factor	1*108	5*109
Amplification curves (n=48)
Derivative of the melting curves (n=48)

The data from the limiting dilution experiment are summarized in the table below

Creating “hot start” TAQ, and test thereof

Use of Taq polymerase specific monoclonal antibodies (Mab) to inhibit enzymatic activity (before initial denaturation in the PCR) is a well-documented method. Antibodies was expressed in hybridomas and purified by David J. Warren. Three different concentrations of Mab/Taq mixes were tested.

Mixes of Taq and Mab made before the setup of the PCR reaction.

Master Mix without Taq

19µl of the different Taq/Mab mixes where added to 250µl of the master mix above. 0.5 µl Taq (without Mab) was added to the control.

Derivative of the melting curve and the amplification curves are depicted below in the following order.

Primer set #21	0 MAb	0,1µg MAb	0,5µg MAb	1µg MAb
Amplification curves
Derivative of the melting curve
Mean Cq	24.4	25.0	25.8	26.1
1 STD	0.12	0.07	0.33	0.57

The data show that there are more impurities with reduced MAb concentration. Which also corresponds with the Cq values. The PCR was started with 2 minute incubation at 94°C. This is most likely not enough to denature all the MAb at the highest concentration which is reflected in a high Cq value (needs a few more cycles to get going). This can also increase the variation between the 24 replicates.

MAb testing with complete one tube chemistry.

Starting template, pool of RNA from Rikshospitalet (anonymous samples) spiked with Covid RNA from Trondheim. First experiment, keeping complete mix at 4°C for 2.5h The mix is split in 2, one subjected to cDNA synthesis 42°C 180 sec and 50°C 180 sec, then store at 4°C for 2.5h, the other half, straight into the fridge. After 2.5 hours, plate out and run cDNA synthesis and TaqMan probe PCR.

	Straight into the fridge, store at 4°C for 2.5h	cDNA synthesis 42°C 180 sec and 50°C 180 sec, then store 4°C for 2.5h
Taq (no Mab)
Taq with 0.5µg MAb
Taq with 1µg MAb

Reproducebility

Starting template, pool of RNA from Rikshospitalet (anonymous samples) spiked with Covid RNA from Trondheim.

48 replicates with Taq plus 0.5µg or 1µg MAb. Blue is covid signal, green internal standard.

0.5µg Mab	1µg Mab

Links to short PDF file with data