CoV-2-KomET – With high-throughput diagnostics of SARS-CoV-2

A prerequisite for the assessment of new infections with SARS-CoV-2 is testing as comprehensively as possible. For current approaches, SARS-CoV-2 nucleic acids are transcribed into DNA molecules using a so-called reverse transcription, multiplied by a quantitative polymerase chain reaction (PCR) and analyzed. The sample throughput of the qRT-PCR devices is limited, as is the availability.

Fraunhofer researchers therefore have developed, validated and tested a nucleic acid‑based high-throughput method to increase the sample throughput by at least one order of magnitude compared to the standard method.

For this novel approach, the researchers take advantage of the high-throughput potential of modern sequencing technology: the various patient samples are not to be analyzed individually, but rather an entire pool – consisting of samples from thousands of patients – is to be sequenced simultaneously.

High-throughput diagnostics of respiratory diseases such as SARS-CoV-2

According to the Robert Koch Institute, approximately 14.75 million SARS-CoV-2 infections have been detected in Germany to date [1], with a higher number of unreported cases suspected. However, the currently used diagnostic methods of reverse transcription of viral nucleic acids followed by quantitative polymerase chain reaction, widely known as PCR tests, are limited in terms of sample throughput. In addition, infections with other seasonal respiratory diseases such as influenza or RSV (respiratory syncytial virus), are also increasing [2], which would need to be analyzed simultaneously in the best possible way.

Procedure of CoV-2-KomET, starting with sample collection to molecular barcoding in RT-PCR, analysis and evaluation of results. Created with Biorender. — Procedure of CoV-2-KomET, starting with sample collection to molecular barcoding in RT-PCR, analysis and evaluation of results. Created with BioRender.

Area-wide testing by high-throughput diagnostics

Therefore, in order to be able to offer area-wide PCR testing with regard to the current Omikron variant, sample throughput must be significantly increased. In collaboration with the Fraunhofer Institutes for Cell Therapy and Immunology IZI, and Production Technology and Automation IPA, we at Fraunhofer IGB have developed a new method for high-throughput diagnostics of SARS-CoV-2 and validated it on clinical samples as part of the Fraunhofer CoV-2-KomET project. This combines reverse transcription and amplification of specific viral sequences (RT-PCR) with the high-throughput capability of modern sequencing technologies. In order to analyze thousands of patients simultaneously, samples are labeled with specific oligonucleotides – also known as “molecular barcodes” – in the RT-PCR step, which allows a unique assignment to the patient (see Fig.).

First validation of the approach

CoV-2-KomET was successfully tested on a total of 672 clinical samples and achieved a specificity of over 89 percent and a sensitivity of over 87 percent in comparison to standard PCR tests. The diagnostic value of CoV-2-KomET, determined by the “area under the curve (AUC)”, of 94 percent also demonstrates the potential of this approach, which will be subject to further evaluation and improvement in the future. In addition to the pure SARS-CoV-2 high-throughput detection, a “respiratory panel” was successfully established on 41 synthetic samples, allowing the detection of SARS-CoV-2 as well as Influenza A/B down to a detection limit of 10 copies/µL.

Outlook

CoV-2-KoMET, as a diagnostic approach based on high-throughput sequencing of viral nucleic acids, represents a promising and alternative method for pandemics and infection outbreaks. Not only could sample throughput be increased with this approach, but scalability could also reduce the potential costs per test. Furthermore, by changing the design of the specific viral sequences, it is possible to respond to new variants within a few weeks. The project will be continued with the industrial partner LMV Laboratories, with a special focus on improving and expanding the respiratory panel to simultaneously test patients for SARS-CoV-2, Influenza and RSV within one sample.

References

[1] RKI Dashboard, as of February 28, 2022

[2] Buda, S.; Dürrwald, R.; Biere, B.; Reiche, J.; Buchholz, U.; Tolksdorf, K.; Schilling, J.; Goerlitz, L.; Streib, V.; Preuß, U.; Prahm, K.; Haas, W. und die AGI-Studiengruppe (2021) ARE-Wochenbericht – Aktuelles zu akuten respiratorischen Erkrankungen Kalenderwoche 47, Robert Koch-Institut

Project information

Project title

Cluster project CoV‑2‑KomET – Detection of SARS‑CoV‑2 with subproject "High‑throughput diagnostics of SARS‑CoV‑2"

Project duration

September 2020 – November 2021

Project partners

Project funding

This work was supported by the Fraunhofer InternaI Programs under Grant No. Anti-Corona 840268 (»Fraunhofer vs. Corona«)