| Autor: |
Eva Krause, Janine Michel, Andreas Puyskens, Natalie Hofmann, Thomas Rinner, Barbara Biere, Brigitte G. Dorner, Martin Skiba, Lars Schaade, Andreas Nitsche |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
Virology Journal, Vol 20, Iss 1, Pp 1-13 (2023) |
| Druh dokumentu: |
article |
| ISSN: |
1743-422X |
| DOI: |
10.1186/s12985-023-02088-x |
| Popis: |
Abstract Background Over the course of the COVID-19 pandemic, laboratories worldwide have been facing an unprecedented increase in demand for PCR testing because of the high importance of diagnostics for prevention and control of virus spread. Moreover, testing demand has been varying considerably over time, depending on the epidemiological situation, rendering efficient resource allocation difficult. Here, we present a scalable workflow which we implemented in our laboratory to increase PCR testing capacity while maintaining high flexibility regarding the number of samples to be processed. Methods We compared the performance of five automated extraction instruments, using dilutions of SARS-CoV-2 cell culture supernatant as well as clinical samples. To increase PCR throughput, we combined the two duplex PCR reactions of our previously published SARS-CoV-2 PCR assay into one quadruplex reaction and compared their limit of detection as well as their performance on the detection of low viral loads in clinical samples. Furthermore, we developed a sample pooling protocol with either two or four samples per pool, combined with a specifically adapted SARS-CoV-2 quadruplex PCR assay, and compared the diagnostic sensitivity of pooled testing and individual testing. Results All tested automated extraction instruments yielded comparable results regarding the subsequent sensitivity of SARS-CoV-2 detection by PCR. While the limit of detection of the quadruplex SARS-CoV-2 PCR assay (E-Gene assay: 28.7 genome equivalents (ge)/reaction, orf1ab assay: 32.0 ge/reaction) was slightly higher than that of our previously published duplex PCR assays (E-Gene assay: 9.8 ge/reaction, orf1ab assay: 6.6 ge/reaction), the rate of correctly identified positive patient samples was comparable for both assays. Sample pooling with optimized downstream quadruplex PCR showed no loss in diagnostic sensitivity compared to individual testing. Conclusion Specific adaptation of PCR assays can help overcome the potential loss of sensitivity due to higher levels of PCR multiplexing or sample dilution in pooled testing. Combining these adapted PCR assays with different sample processing strategies provides a simple and highly adjustable workflow for resource-efficient SARS-CoV-2 diagnostics. The presented principles can easily be adopted in a variety of laboratory settings as well as be adapted to pathogens other than SARS-CoV-2, making it feasible for any laboratory that conducts PCR diagnostics. |
| Databáze: |
Directory of Open Access Journals |
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