Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2

Abstract

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.

Figure 1:. The SARS-CoV-2 ORF1ab and N gene LAMP primer sets are specific.

Only samples with SARS-CoV-2 produced a positive signal, while negative controls (PEDV, TGE, PDCoV, IBV, MERS-CoV, and SARS-CoV) did not show any amplification signal. 10⁴ copies of coronaviruses genome RNAs (PEDV, TGE, PDCoV, IBV), cDNA (MERS-CoV), or synthetic DNA (SARS-CoV) were added to each reaction. Reverse transcriptase (Promega) was included in the LAMP reaction mix.

Figure 2:. Real Time and Colorimetric detection of SARS-CoV-2.

(A) and (B) Visual end point detection (LCV dye) of SARS-CoV-2 amplicons with our direct COVID-19 RT-LAMP and our Closed-Tube Penn-RAMP with ORF1ab and N gene LAMP primer sets, respectively. (C) and (D) are the LAMP amplification curves corresponding to RT LAMP in (A) and (B). (E) and (F) The LAMP threshold time in (C) and (D) as a function of SARS-CoV-2 concentration (n = 3).

Figure 3:. Inhibition effect of swab sample collection medium and saliva on direct RT-LAMP.

(A) The impact of VTM, saline, water, and saliva on colorimetric detection of SARS-CoV-2 with LCV dye in the presence and absence of TCEP/EDTA and/or heat treatment. (B) and (C) The LAMP threshold time as a function of medium type in the presence (B) and the absence of heat treatment (C) (n = 3). (D) Colorimetric detection of SARS-CoV-2 after storing the samples at 4°C for a week. (E) The LAMP threshold time in (D) as a function of medium type and heat treatment (n = 3). RT = room temperature. All LAMP experiment was carried out with OptiGene master mix (ISO-001) and N gene LAMP primer set.

Figure 4:. Saliva sample detection.

(A) Colorimetric detection of the SARS-CoV-2 virus in contrived samples comprised of virions spiked in saliva. (B) RT-LAMP threshold time as a function of the SARS-CoV-2 virus concentration (n = 3). (C) Colorimetric detection of SARS-CoV-2 in saliva samples from suspected COVID-19 patients. A 4 μL of saliva was directly added to RT-LAMP reaction mix. (D) RT-PCR amplification curves of the saliva samples in (C).

Figure 5:. Block heater and dried reagents.

(A), (B), and (C) outside, inside, and exploded views of our block heater. (D) Colorimetric detection of SARS-CoV-2 with dried reaction mix and LCV dye. 10³ SARS-CoV-2 virions used in the positive control. (E) Colorimetric detection of SARS-CoV-2 with dry LAMP reaction mixture in the presence of 10⁵, 10⁴, 10³, 100, 10, and 0 virions per reaction. These reactions were incubated with our custom-made block heater.