Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

Abstract

The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

Figure 1

An RT-LAMP assay for rapid and direct SARS-Cov-2 testing of clinical sample and the point-of-care. (A) Primer design. A set of 6 LAMP primers targeting the middle of the Orf1ab gene. Sequences and primers matching to the + strand of virus genome are in pink, while those matching to the—strand are shown in blue. Illustration of GC% of SARS-CoV-2 genome was from UCSC genome browser (http://genome.ucsc.edu)^,. (B) Workflow of direct RT-LAMP assay. 20 µL of raw clinical samples from nasal swab in viral transport media (VTM) was added to 480 µL reaction solution consisting of reaction master mix and lysis buffer, mixed and 250 µL were aliquoted with a transfer pipette and then incubated on heatblock for 30 min. The reaction was stopped by placing the samples on ice and results were interpreted by color due to a pH sensitive dye in the master mix (yellow = positive; red = negative). (C). Setup of the RT-LAMP assay. (D). An example of colorimetric results on negative and positive clinical samples.

Figure 2

The performance of RT-LAMP testing for SARS-Cov-2. (A) Estimation of limit of detection using VTM and RNA standard spike-ins. (Colorimetric results: red = negative; yellow = positive). (B) Estimation of limit of detection using clinical samples selected to represent a wide range of Ct values. Each dot represents the Ct value of target 2 of one sample, and a sample with discordant testing result is labeled in red. Error bar indicates Mean ± SD. (C) Testing on randomly selected positive and negative clinical samples. Each dot represents the Ct value of target 2 of one sample, and a sample with discordant testing result is labeled in red. Error bars indicates mean ± SD.