tetano
Editor, Senior Moderator
PLoS One
. 2020 Nov 2;15(11):e0238612.
doi: 10.1371/journal.pone.0238612. eCollection 2020.
Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay
Bernhard Schermer[SUP] 1 2 3 [/SUP], Francesca Fabretti[SUP] 1 2 3 [/SUP], Maximilian Damagnez[SUP] 4 [/SUP], Veronica Di Cristanziano[SUP] 4 [/SUP], Eva Heger[SUP] 4 [/SUP], Sita Arjune[SUP] 1 2 3 [/SUP], Nathan A Tanner[SUP] 5 [/SUP], Thomas Imhof[SUP] 2 6 [/SUP], Manuel Koch[SUP] 2 6 [/SUP], Alim Ladha[SUP] 7 8 9 10 [/SUP], Julia Joung[SUP] 7 8 9 10 [/SUP], Jonathan S Gootenberg[SUP] 9 11 [/SUP], Omar O Abudayyeh[SUP] 9 11 [/SUP], Volker Burst[SUP] 1 12 [/SUP], Feng Zhang[SUP] 7 8 9 10 11 13 [/SUP], Florian Klein[SUP] 2 4 14 [/SUP], Thomas Benzing[SUP] 1 2 3 [/SUP], Roman-Ulrich M?ller[SUP] 1 2 3 [/SUP]
Affiliations
Abstract
Background: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.
Methods: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).
Results: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.
Conclusion: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.
. 2020 Nov 2;15(11):e0238612.
doi: 10.1371/journal.pone.0238612. eCollection 2020.
Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay
Bernhard Schermer[SUP] 1 2 3 [/SUP], Francesca Fabretti[SUP] 1 2 3 [/SUP], Maximilian Damagnez[SUP] 4 [/SUP], Veronica Di Cristanziano[SUP] 4 [/SUP], Eva Heger[SUP] 4 [/SUP], Sita Arjune[SUP] 1 2 3 [/SUP], Nathan A Tanner[SUP] 5 [/SUP], Thomas Imhof[SUP] 2 6 [/SUP], Manuel Koch[SUP] 2 6 [/SUP], Alim Ladha[SUP] 7 8 9 10 [/SUP], Julia Joung[SUP] 7 8 9 10 [/SUP], Jonathan S Gootenberg[SUP] 9 11 [/SUP], Omar O Abudayyeh[SUP] 9 11 [/SUP], Volker Burst[SUP] 1 12 [/SUP], Feng Zhang[SUP] 7 8 9 10 11 13 [/SUP], Florian Klein[SUP] 2 4 14 [/SUP], Thomas Benzing[SUP] 1 2 3 [/SUP], Roman-Ulrich M?ller[SUP] 1 2 3 [/SUP]
Affiliations
- PMID: 33137122
- DOI: 10.1371/journal.pone.0238612
Abstract
Background: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.
Methods: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).
Results: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.
Conclusion: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.