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

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.

Fig 1. Detection of viral RNA with RT-LAMP.

A. Representative pictures of a RT-LAMP assay. UTM samples from 6 patients, 4 of which tested positively for SARS-CoV-2 in diagnostic qPCR were analyzed by RT-LAMP targeting Gene N and Orf1a. Isolated RNA from swabs served as positive and negative control. B. 70 samples were analyzed with three different RT-LAMP assays targeting Gene N, Orf1a and Orf7a. Representative results from additional assays. C. The individual value plot shows of Ct values of the diagnostic qPCR for the E gene of positive and negative RT-LAMP assays. D. Total number of positive and negative RT-LAMP assays from patient samples tested positive or negative for SARS-CoV-2. E. Heat map summarizing the results of three different RT-LAMP assays on all 70 samples (left column: Ct value for E gene from diagnostic PCR in ascending order; other columns: pink indicating positive, blue indicating negative result. Grey: not performed).

Fig 2. Detection of viral RNA with multiplexed RT-LAMP.

A. Workflow of the optimized multiplexed RT-LAMP protocol. B. 104 samples were analyzed with the multiplexed RT-LAMP assay targeting Orf7a and Orf3a. The individual value plot shows Ct values of the diagnostic qPCR for the E gene of positive and negative RT-LAMP assays. C. Total number of positive and negative multiplexed RT-LAMP assays from patient samples tested positive or negative for SARS-CoV-2. D. Heat map summarizing the results (left column: Ct value for E gene from diagnostic PCR in ascending order; middle and right column: pink indicating positive, blue indicating negative result. Grey: not performed).