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.

Keywords: RT-LAMP; SARS-CoV-2; diagnostic.

Fig. 1.

Sequence conservation of Orf1a-HMS target region between related coronaviruses and within SARS-CoV-2 isolates. (A) An alignment (blastn, megablast) of SARS, Bat SARS-like coronavirus isolate Rs4084, and the sequence detected by Orf1a-HMS/Orf1a-HMSe. (B and C) Measure of sequenced nucleotide mutations in a subset of global data provided by Nextstrain (nucleotide entropy), adapted from Nextstrain visual interface. Orf1a-HMS/Orf1a-HMSe target region (2245 to 2441) indicated. (B) Whole genome view. (C) Close-up of the target region.

Fig. 2.

Initial sensitivity test of selected RT-LAMP primer sets. Fluorescent RT-LAMP reactions run for 120 30-s cycles at 65 °C: 0 (blue), 100 (green), 200 (red), or 300 (purple) control RNA copies included per reaction (n = 4). Assays performed were (A) Orf1a-HMS and (B) Orf1a-HMSe. RFU, relative fluorescence units.

Fig. 3.

Assessment of RT-LAMP detergent tolerance. Fluorescent RT-LAMP reactions run for 120 30-s cycles at 65 °C. All reactions contain 500 control RNA copies, the Orf1a-HMS primer set, and 0 to 3% added (A) Tween20 or (B) TritonX100, as indicated.

Fig. 4.

Assessment of GuSCN effects on sensitivity of colorimetric RT-LAMP assays. Colorimetric RT-LAMP reactions run with the number of control RNA copies (0, 100, or 200) noted. Reactions were run with 50 mM GuSCN (+) or without GuSCN (−) as noted. (A) NEB Orf1a-C primer set. (B) NEB Gene N-A primer set. (C) Orf1a-HMS primer set. (D) Orf1a-HMSe primer set. An * indicates reactions that were noticeably orange, but not completely yellow.

Fig. 5.

A simple and rapid sample inactivation and purification scheme. (A) A schematic depicting sample inactivation and direct RT-LAMP testing. (B) A schematic of the purification and testing procedure for swab samples postinactivation. (C) A schematic of the purification and testing procedure for saliva samples postinactivation.

Fig. 6.

Sensitivity test with Orf1a-HMSe primer set following sample inactivation. Direct detection in reconstituted throat and nasal swabs in (A) saline, (B) 1× PBS, or (C) reconstituted saliva. Control RNA copies were spiked into samples during inactivation (concentration indicated). Negative control samples (−) had no RNA added. Following inactivation, 5 μL of sample was added to a colorimetric RT-LAMP reaction with Orf1a-HMSe primers. Positive control reactions (+) had an additional 1,000 control RNA copies added directly to the reaction.

Fig. 7.

Sensitivity test with Orf1a-HMSe primer set following sample purification. Direct detection in reconstituted throat and nasal swabs in (A) saline, (B) 1× PBS, or (C) reconstituted saliva. Control RNA copies were spiked into samples during inactivation (concentration indicated). Negative control samples (−) had no RNA added. Following inactivation, samples were purified using glass milk with a centrifuge, and colorimetric RT-LAMP reaction with Orf1a-HMSe primers was added directly. Positive control reactions (+) had an additional 1,000 control RNA copies added directly to the reaction. Reactions were run for 30 min to 40 min at 65 °C, as indicated.

Fig. 8.

Evidence for RNA stability from inactivation through purification. (A) Swabs in saline, swabs in 1× PBS, saliva, or a 1:1 mixture of saliva and saline was inactivated and spiked with control RNA to 100 copies per microliter. After inactivation, samples were incubated for 30 min at 37 °C for 30 min or 24 h at room temperature, as indicated, before 5 μL was added to colorimetric RT-LAMP reaction mix with Orf1a-HMSe primers. (B) The 500-μL swabs in saline were purified using glass milk with a centrifuge. Dried RNA/silica pellets were left at room temperature for 48 h before adding colorimetric RT-LAMP reaction mix with Orf1a-HMSe primers. Negative control sample (−) had no RNA copies added to sample. Positive control reactions (+) had 1,000 RNA copies spiked directly into the reaction. (C and D) RNase activity from swabs placed directly into NaI solutions (concentration indicated). RNase Alert reactions incubated for 30 min at 37 °C; (C) bright field, (D) 488 nm fluorescence channel; fluorescence indicates RNase activity.