SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

Abstract

As the current SARS-CoV-2 pandemic spreads, the need for more diagnostic capabilities is great. In order to address this need, we have developed a highly sensitive RT-LAMP assay compatible with current reagents, that utilizes a colorimetric readout in as little as 30 minutes. In addition to this, we have developed an inexpensive pipeline to further increase sensitivity without requiring highly specialized equipment. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was also developed to increase sensitivity and sample stability. This protocol, combined with our 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 our RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. We hope that this inactivation and purification pipeline, which costs approximately $0.07 per sample and which uses readily available reagents, will increase the availability of SARS-CoV-2 testing, as well as expand the settings in which this testing can be performed.

Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification

“…While this clinical validation is focused on nasopharyngeal swabs, which are recommended by the U.S. Centers for Disease Control as the most sensitive specimen type for SARS-CoV-2 detection [11], the same methods can be applied to other sample types as well, possibly including saliva [8]. Oropharyngeal specimens are likely to be compatible given their similar composition to nasopharyngeal specimens.…”

“…4A). We used the glass milk protocol developed by Rabe and Cepko [8] to concentrate up to 500 µL of sample into a single RT-LAMP reaction. Purification improved the LoD by ten-fold ( Fig.…”

“…Regardless of the sample preparation method, each sample was amplified with two SARS-CoV-2-specific primer sets for the ORF1a gene [8] and N gene [9]. An additional primer set for the human actin B gene also served as an internal specimen control to detect the presence of inhibitors.…”

Screening for SARS-CoV-2 infections with colorimetric RT-LAMP and LAMP sequencing

“…However, Proteinase K has the same temperature optimum as the RT-LAMP reaction (65°C) and heat inactivation is not 100% efficient, even when incubating at 95°C for 10 min (see FAQ pages on www.qiagen.com), thus exposing the enzymes in the RT-LAMP reaction to unpredictable proteolytic influences. Rabe and Cepko (2020) suggest to use cheap silika preparation and novel sample inactivation protocols to enrich the RNA prior to LAMP, which however would come at the expense of complicating the simple swab-to-RT-LAMP workflow. Finally, our analysis found that a short heat treatment of 5 min at 95 °C, which poses minimal additional handling steps, does not destroy the RNA but rather stabilised it in the inactivated specimen, and improved the sensitivity and specificity of the swab-to-RT-LAMP assay (Figure 8).…”

“…One promising lead for future applications is the exploration of hot swab-to-RT-LAMP with saliva specimens, which may show a higher sensitivity than in oropharyngeal swab specimens Wyllie et al, 2020). Compatibility of RT-LAMP with direct saliva specimens has been shown before but only using spiked-in IVT RNA (Badhra et al, 2020, Rabe andCepko 2020).…”