Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic
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 o...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2023-07-01
|
| Series: | Virology Journal |
| Online Access: | https://doi.org/10.1186/s12985-023-02088-x |
| _version_ | 1797784749819822080 |
|---|---|
| author | Eva Krause Janine Michel Andreas Puyskens Natalie Hofmann Thomas Rinner Barbara Biere Brigitte G. Dorner Martin Skiba Lars Schaade Andreas Nitsche |
| author_facet | Eva Krause Janine Michel Andreas Puyskens Natalie Hofmann Thomas Rinner Barbara Biere Brigitte G. Dorner Martin Skiba Lars Schaade Andreas Nitsche |
| author_sort | Eva Krause |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T00:44:20Z |
| format | Article |
| id | doaj.art-3bb695e9e95840b8acb6063f9127768f |
| institution | Directory Open Access Journal |
| issn | 1743-422X |
| language | English |
| last_indexed | 2024-03-13T00:44:20Z |
| publishDate | 2023-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Virology Journal |
| spelling | doaj.art-3bb695e9e95840b8acb6063f9127768f2023-07-09T11:05:59ZengBMCVirology Journal1743-422X2023-07-0120111310.1186/s12985-023-02088-xFlexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemicEva Krause0Janine Michel1Andreas Puyskens2Natalie Hofmann3Thomas Rinner4Barbara Biere5Brigitte G. Dorner6Martin Skiba7Lars Schaade8Andreas Nitsche9Centre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteDepartment for Infectious Diseases, Unit Influenza and Other Respiratory Viruses (FG 17), Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Biological Toxins (ZBS 3), WHO Collaborating Centre for Emerging Infections and Biological Threats, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Biological Toxins (ZBS 3), WHO Collaborating Centre for Emerging Infections and Biological Threats, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteCentre for Biological Threats and Special Pathogens, Unit Highly Pathogenic Viruses (ZBS 1), WHO Collaborating Centre for Emerging Infections and Biological Threats, WHO Reference Laboratory for SARS-CoV-2, Robert Koch InstituteAbstract 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.https://doi.org/10.1186/s12985-023-02088-x |
| spellingShingle | Eva Krause Janine Michel Andreas Puyskens Natalie Hofmann Thomas Rinner Barbara Biere Brigitte G. Dorner Martin Skiba Lars Schaade Andreas Nitsche Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic Virology Journal |
| title | Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic |
| title_full | Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic |
| title_fullStr | Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic |
| title_full_unstemmed | Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic |
| title_short | Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic |
| title_sort | flexible upscaling of laboratory pcr testing capacity at the robert koch institute during the sars cov 2 pandemic |
| url | https://doi.org/10.1186/s12985-023-02088-x |
| work_keys_str_mv | AT evakrause flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT janinemichel flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT andreaspuyskens flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT nataliehofmann flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT thomasrinner flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT barbarabiere flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT brigittegdorner flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT martinskiba flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT larsschaade flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic AT andreasnitsche flexibleupscalingoflaboratorypcrtestingcapacityattherobertkochinstituteduringthesarscov2pandemic |