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Kilobaser Application Note:

Covid-19 Primer

Comparison of Kilobaser and Eurofins Primer

Summary

How is the performance of primers synthesized by Kilobaser? The stepwise yield of primers made by Kilobaser is about 99.5%, which is comparable to any other manufacturer. However, to demonstrate the performance of Kilobaser primers in an experiment, COVID-19 primers synthesized by Kilobaser were compared to primers purchased from Eurofins. In order to do that, 2019-nCoV_N1 forward and reverse primer were tested in a real time quantitative PCR (qPCR). The Cq values, the result you get from the qPCR test, indicates that primers from Kilobaser and Eurofins show an equal performance.

Introduction

Covid-19

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to more than 17 million confirmed cases with over 680,222 deaths worldwide, as of July 31, 2020. More than 11 million already recovered but five million are still infected at this moment, of which over 65K show serious symptoms. Billions of people on this planet live and work in disrupted conditions due to different forms of regulations for social distancing and lockdowns. Supply chains are disrupted or even stopped due to business shutdowns and major restrictions in travel.

Due to that oligo shipment is slowed down as well, especially in a time, where not only Covid-19 primers but also any other oligos are highly needed. DNA sequences are involved in the development of PCR tests for diagnosing the virus faster, in COVID-19 research and in developing vaccines against it.

How can Kilobaser be helpful during Covid-19 pandemic?

Kilobaser is a benchtop DNA synthesizer that connects a microfluidic chip technology and a single reagent cartridge.

Due to long waiting times for primers as supply chains are disrupted during COVID-19 pandemic Kilobaser can accelerate the development of efficient testing methods for the detection of SARS-CoV-2. Additionally, it can help to accelerate COVID-19 research and vaccine development.

What is the principle of qPCR?

Nucleic acid amplification and detection techniques are used in a broad range of applications in all areas of life science – basic research, medicine, diagnostics, biotechnology, forensics and more.

In traditional PCR an amplified DNA product is detected in an end-point method. In qPCR the accumulation of DNA is monitored in real time as the reaction progresses during amplification. This can be done using a sequence-specific DNA probe with a fluorophore and a quencher or a fluorescent dye. In this experiment a fluorescent dye is used.

Real-time PCR connects amplification and detection into one single step. It can be used for qualitative (the presence or absence of a sequence) and quantitative (copy number) analysis. Within this experiment, a fluorescent dye is used to label the PCR product. The PCR instrument can monitor the accumulation of fluorescent signal during the exponential phase of the reaction for precise, sensitive and quantitative analysis of the PCR product. The measured fluorescence is proportional to the total amount of amplified DNA product or also called amplicon.

Why is the cq value?

Initially, the fluorescent signal, is too low to be detected, even though product accumulates. Eventually, when enough product is amplified, the fluorescent signal is detectable. The cycle number at which fluorescence can be detected is the basic result of qPCR and is called Quantification Cycle (Cq for short).

For example, low Cq values represent higher initial amount of the target DNA. Sometimes Cq values are also called Ct (threshold cycle) values or Cp (crossing point) values in literature. However, there is no difference in the meaning of these values. “Cq or Quantification cycle” is the correct naming according the MIQE guidelines as described in Bustin et al.; Clinical Chemistry 55:4; 2009.

Material and Methods

To compare the performance of Covid-19 primers from Kilobaser and Online Synthesis provider, Cq values measured by the qPCR instrument (ChaiBio) were compared to each other. This was done by ordering the 2019-nCoV_N1 Forward and Reverse Primers, the positive control, and a qPCR Mix, while the same forward (20mer) and reverse (24mer) primers were printed with Kilobaser. After an automatic final cleavage/deprotection step was performed by the Kilobaser, DNA was eluted dry and ready-to-use.

In the next step DNA was diluted in Tris-EDTA (TE) buffer. The buffer keeps the pH neutral and EDTA, acting as a chelator of cations such as magnesium, protects DNA against enzymatic degradation. After quantifying DNA using a fluorometer, the overall yield of the DNA product can be calculated. The overall yield of oligos made by Kilobaser is between 200 and 300 pmol. The stepwise yield of Kilobaser oligos is between 99.0 and 99.5%.

PCR reaction

2019-nCoV_N1 targets virus nucleocapsid (N protein) gene of SARS-CoV-2. The reason for targeting this region is that it is a highly conserved region in coronaviruses and likely to show a consistent result as the pandemic continues. The qPCR Mastermix, 5x Hot Firepol EvaGreen qPCR Mix Plus no ROX purchased from Solis BioDyne contains a fluorescent dye, a DNA polymerase and dNTPs. Additionally, forward and reverse primers with a concentration of 10 pmol from Eurofins or Kilobaser and the template DNA purchased from IDT (positive control) were added to the qPCR mix.

Primer Label (Nucleotide sequence 5´-3´)

2019-nCoV_N1 Forward Primer (GAC CCC AAA ATC AGC GAA AT)

2019-nCoV_N1 Reverse Primer (TCT GGT TAC TGC CAG TTG AAT CTG)

Mastermix

Forward Primer (~10pmol) (Eurofins or Kilobaser)

Reverse Primer (~10pmol) (Eurofins or Kilobaser)

1µl Template DNA (2019-nCoV_N Positive Control (IDT) (200.000 copies/µl) (IDT)

5x Hot Firepol EvaGreen qPCR Mix Plus no ROX (Solis BioDyne)

Water

 

Thermal cycling profile

DNA is amplified in a typical qPCR run creating repeated cycles of alternating temperature periods. The thermal cycling profile is illustrated in figure 1. This reaction was run for 40 cycles. For more details about each step have a look at one of our previous blogposts, PCR, COVID-19 and Kilobaser.

 

Figure 1: Thermal cycling profile for qPCR (ChaiBio).

Figure 1: Thermal cycling profile (ChaiBio) for qPCR. Reaction was run for 40 cycles.

Results

Figure 2 illustrates a typical amplification plot with different amplification curves. In this plot, the x-axis shows the number of PCR cycles, and the fluorescence from the amplification reaction, which is proportional to the amount of amplicon in the tube, is shown on the y-axis.

 

The purple amplification curve shows a reaction where Eurofins forward and Kilobaser reverse primer were used. The pink curve indicates the use of Eurofins forward and reverse primer. The dark blue curve represents the reaction of Kilobaser forward and Eurofins reverse primer and the light blue curve shows the use of forward and reverse primer made by Kilobaser. The second values of the double determination were not shown due to clarity/illustration reasons.

The Quantifiation cycle (Cq) value represents the initial amount of template present at the start of the amplification reaction. For example, low Cq values represent higher initial amount of the target DNA.

The Cq values vary from 24.39 to 23.47, which means they are all in the same range. The Cq value of the curve where only Kilobaser primers were used is 24.39; the Cq value of the reaction where Eurofins primers were used, is smaller by the value of 0.39.

Figure 1: qPCR amplification curves performed by ChaiBio qPCR thermocycler. X-axis shows the cycle number and the y-axis shows the relative fluorescence units. Purple curve: forward primer (Eurofins), reverse primer (Kilobaser).  Pink curve: forward primer (Eurofins), reverse primer (Eurofins). Dark blue curve: forward primer (Kilobaser), reverse primer (Eurofins). Light blue: forward primer (Kilobaser), reverse primer (Kilobaser).

Figure 2: qPCR amplification curves performed by ChaiBio qPCR thermocycler. X-axis shows the cycle number and the y-axis shows the relative fluorescence units. Purple curve: forward primer (Eurofins), reverse primer (Kilobaser). Pink curve: forward primer (Eurofins), reverse primer (Eurofins). Dark blue curve: forward primer (Kilobaser), reverse primer (Eurofins). Light blue: forward primer (Kilobaser), reverse primer (Kilobaser). Baseline substraction is active, meaning that the backround signal is substracted.

Conclusion

Comparing the Cq values resulting from the qPCR amplification, primers made by Kilobaser show the same performance as primers purchased from online synthesis services. Kilobaser seems to be a useful and time-saving add-on for PCR systems, as constant primer supply is given. Due to the low reaction volume in the microfluidic chip, the salt concentration is kept exceptionally low. Thus, no additional desalting step is needed after DNA synthesis. The total liquid volume in reagent cartridges is 15 ml, hence 50% less hazardous waste is produced compared to usual DNA synthesizers.

 

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