SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

Abstract

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

“…Next, we compared the Gene-N-A, Color-N, Color-E, and Color-N/E combination to second-generation primers described in Zhang et al [22] (Gene-N2, Gene-E1 and As1e). We found that As1e, originally published by Rabe et al [29], yielded the lowest Cq value followed closely by a combination of As1e with the two primers targeting the Gene-N2 and Gene-E1 genes designed by Zhang et al (Fig 3B). The Color-N primer yielded similar Cq values to the triple combination primer set from Zhang et al We also tested the Color-N, Color-E1, and Gene-N-A gene primer sets against additional published primers that target ORF1a (Lamb, Yu, El-Tholoth, and Zhang primers) either alone or in combination and compared them to the Gene-N-A gene primer set (S1 Table).…”

“…We used fluorescent-based detection with Warmstart LAMP reagents and the included fluorescent dye (New England Biolabs, NEB cat # E1700L). We tested primer sets developed in previous studies targeting several SARS-CoV-2 genes as shown in S1 Table [13,14,22,[29][30][31][32][33]. Of note, the Color-Orf1a primers and Lamb-Orf1a primers are identical but were used at different concentrations per the protocols developed by each originating lab.…”

“…The specificity of the primers tested in this manuscript were established in previous publications [13,14,22,29,[31][32][33]. Briefly, the Gene-N-A primers, used in most of the experiments in this manuscript, did not cross-react with SARS-CoV-1 N-gene by RT-LAMP and were not anticipated to cross-react with other common respiratory pathogens based on sequence comparisons (communication with Nathan Tanner).…”

An enhanced isothermal amplification assay for viral detection

“…RPA has potential advantages over other isothermal amplification technologies such as loop-mediated isothermal amplification (LAMP) 13 as it can be performed near ambient temperature (37-42°C) and is more rapid. While several creative applications of LAMP technologies to diagnose COVID-19, the disease caused by SARS-CoV-2, have recently been developed and show promise [14][15][16][17][18] , RT-RPA has been less explored.…”

Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes

“…We envisioned jLAMP assay workflow as displayed in Figure 3a, where heat treated swab media or saliva is directly added to the jLAMP master mix, heated at 60 °C and read out visually. First, we screened several sets of LAMP primers [8][9][10] to achieve optimum performance and found that those designed by D. Wang 10 yielded the best sensitivity. We also designed primers specific for the human RPP30 and 18S rRNA genes to be used to test sample integrity.…”