Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay

PLoS One. 2020 Nov 2;15(11):e0238612. doi: 10.1371/journal.pone.0238612. eCollection 2020.

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.

MeSH terms

  • Betacoronavirus / genetics*
  • Betacoronavirus / isolation & purification
  • COVID-19
  • COVID-19 Testing
  • Clinical Laboratory Techniques
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • Humans
  • Nasopharynx / virology
  • Nucleic Acid Amplification Techniques / methods*
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*
  • Real-Time Polymerase Chain Reaction
  • SARS-CoV-2
  • Sensitivity and Specificity

Substances

  • RNA, Viral

Grant support

F.Z., O.O.A., J.S.G., J.J., and A.L. are inventors on patent applications related to SHERLOCK technology filed by the Broad Institute, with the specific aim of ensuring this technology can be made freely, widely, and rapidly available for research and deployment (US provisional application no. 62/975,743, "CRISPR Effector System Based Coronavirus Diagnostics"). This does not alter our adherence to PLOS ONE`s policies on sharing data and materials. O.O.A., J.S.G., and F.Z. are co-founders, scientific advisors, and hold equity interests in Sherlock Biosciences, Inc. F.Z. is also a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, and Arbor Biotechnologies. NAT is employed by New England Biolabs, Inc, manufacturer of the LAMP reagents and some additional enzymes described in this manuscript. All of the aforementioned does not alter our adherence to PLOS ONE`s policies on sharing data and materials. The remaining authors declare no competing financial interests.