A S imple, A ffordable, R apid, S tabilized, Co lorimetric, V ersatile RT-LAMP Assay to Detect SARS-CoV-2

Abstract

The SARS-CoV-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). reverse-transcription (RT)-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high-technological, infrastructure-dependent, and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as an alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step), affordable (~1.7 €/sample), and rapid detection of SARS-CoV-2 targeting both ORF1ab and N genes of the novel virus genome. We demonstrate the assay on RT-qPCR-positive clinical samples, obtaining most positive results under 25 min. In addition, a novel 30-min one-step drying protocol has been developed to stabilize the RT-LAMP reaction mixtures, allowing them to be stored at room temperature functionally for up to two months, as predicted by the Q₁₀. This Dry-RT-LAMP methodology is suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, it could be easily applied both in developed and in low-income countries yielding rapid and reliable results.

Keywords: RT-LAMP; SARS-CoV-2; dry-RT-LAMP; molecular diagnostics; point-of-care.

Figure 1

Schematic representation of COVID-LAMP target localization within SARS-CoV-2 genome. Genbank sequence accession number: MN908947.3 [35].

Figure 2

Real-time RT-LAMP assays performed using the eight primer sets evaluated for the detection of SARS-CoV-2. EvaGreen 20× fluorescence signal over time for primer sets ORF1ab, ORF1b, S447, S555, E, M, N5, and N15 is shown. Orange lines (C+, positive control); black lines (NTC, non-template control). Time to positivity with standard error (Tp (±SE); min) and melting temperatures (Tm; °C) for each primer set are indicated. All reactions were performed in duplicates.

Figure 3

Sensitivity assessment of the RT-LAMP assays for SARS-CoV-2 RNA detection using primer sets ORF1ab, E, N5, and N15. The 10-fold dilutions (1×-1:10⁴) of positive control (C+) are represented by different color lines; non-template control (NTC) is represented by black lines.

Figure 4

Specificity assessment of the RT-LAMP assays for SARS-CoV-2 RNA detection using primer sets ORF1ab, E, N5, and N15. A panel of 13 purified RNA isolates of related viruses obtained from infected patients are included: Coronavirus NL63, Coronavirus OC43, Bocavirus, Rinovirus, Metapneumovirus, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, Enterovirus, Parainfluenzae 1, Influenza H1N1, Influenza A H3, Influenza A H1, and Influenza B. One sample contained RNA from two viruses (Influenza B + Coronavirus OC43); other sample contained RNA from three viruses (Influenza A H1 + Coronavirus OC43 + Adenovirus).

Figure 5

Conventional colorimetric RT-LAMP assays using the primer sets ORF1ab, N5, and N15. Eight RNA isolates from COVID-19 patients (samples nos. 4, 5, 6, 7, 12, 14, 15, and 16) were analyzed by colorimetric RT-LAMP using SYBR Green I fluorescent dye. Green (positive samples), orange (negative samples). C+, RNA-positive control; NTC, non-template control.

Figure 6

Amplification time of Dry-RT-LAMP assays as a function of storage time and temperature. Amplification times of C+ in RT-LAMP assays performed with dry reagents including primer sets ORF1ab, N5, and N15 tested at 0, 1, 7, 14, 21, and 28-days post-desiccation is shown. The different storage temperature (25 °C, 37 °C and 45 °C) is also indicated.

Figure 7

Dry-RT-LAMP assessment using primers set N15 in RNA isolates from COVID-19 patients. Amplification times of samples (S) nos. 4, 5, 6, 7, 12, 14, 15, and 16, performed with dry reagents including primer set N15 at 0, 1, and 7-days post-desiccation are shown. Blue bars (fresh) indicate amplification times obtained in N15-RT-LAMP assays using non-dried reactions (fresh liquid mixes) as reference for comparison. All those reactions were singles.