Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples

Abstract

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

Figure 1

Optimization of enzyme amount and buffer composition for RT-LAMP. All experiments shown were set up with iLACO primers and run for 1 h at 65 °C in a thermocycler, tracked by either SYBR Green I (Bst LF, v5.9) or Eva Green (v7.16) fluorescence. (A) Example of RT-LAMP optimization varying Bst LF amount and the KCl concentration. Positive control synthetic RNA (83,000 copies) and no template control (each in duplicate) were assayed. (B) Summary of optimization of enzyme amount and KCl concentration for Bst LF, v5.9, and v.7.16. Numbers indicate the time difference between the slowest-amplifying positive control replicate and the fastest-amplifying negative control replicate. Color indicates the required time to detect the slowest-amplifying positive control. See Fig. S1 for a additional details regarding how tables are contructed. “ > …” indicates that the negative control did not amplify within 1 h. ND indicates no amplification of either positive or negative control. Two batches of v5.9 with differing activity were assayed, and batch 2 was used for further experiments. (C–D) Optimal conditions determined for v5.9 (0.03 µg/µl in ThermoPol buffer, 10 mM KCl) and v7.16 (0.025 µg/µl in isothermal amplification buffer, 50 mM KCl). 83,000 and 830 copies of RNA were used with v5.9, 10,000 and 1000 with v7.16.

Figure 2

Compatibility of v5.9 and v7.16 polymerases with non-commercial thermostable RT enzymes. All experiments shown were set up with iLACO primers and run for 1 h at 65 °C in a thermocycler, tracked by Eva Green fluorescence. (A) Example amplification plots showing the performance of v5.9 with low or high amounts of RTX (duplicates of 83,000, 8300, and 830 copies of synthetic RNA as well as non template control (NTC)). (B) Optimal detection of synthetic SARS-CoV-2 RNA using v5.9 (0.03 µg/µl in ThermoPol buffer) or v7.16 (0.025 µg/µl in IA buffer) supplemented with MashUp-RT as the thermostable reverse transcriptase. Reactions were performed in triplicate. (C) Optimization of MashUp-RT enzyme amount in combination with v5.9 and v7.16 in their respective optimal conditions (see above for B). 10,000 copies of synthetic RNA and NTC were assayed. Time between positive and negative amplification is indicated in minutes and the color indicates time for detection of the positive controls, as in Fig. 1B.

Figure 3

Benchmarking of in-house produced enzymes against commercial alternatives. Synthetic RNA templates (iLACO and As1e) amplified with the corresponding primers and either WarmStart, Bst3.0 with SSIV, Saphir Bst2.0 Turbo with SSIV, v5.9 with MashUp-RT, or v7.16 with MashUp-RT (same conditions for v5.9 and v7.16 as in Fig. 2B). All experiments were run for 1 h at 65 °C in a thermocycler, tracked by Eva Green fluorescence.

Figure 4

Applicability of RT-LAMP to unextracted nasopharyngeal samples. (A) Effect of common virus transport media on RT-LAMP amplification with v7.16 and MashUp-RT (same conditions and same experiment as in Fig. 2B). Different transport media were added at 10% of the reaction volume. (B) Comparisons of RT-LAMP Ct (minutes) and GeneXpert RT-qPCR Ct (cycles) for 184 clinical samples. The developed v7.16 + RT-MashUP reaction mix was tested with iLACO (red) or As1e primer sets (blue/yellow). As1e primer set was also tested with commercial WarmStart Colorimetric master mix (violet). ND designates the thresholds for calling positives (see methods). (C) Reaction sensitivity according to SARS-CoV-2 abundance as determined by GeneXpert. When technical replicates of As1e were performed and considered together, sensitivity improved (in green, positives were called when at least one replicate was identified as positive). Number of samples in each Ct range: Ct 0–20 = 21, Ct 20–25 = 26, Ct 25–30 = 27, Ct 30–45 = 68. Wilson’s binomial confidence intervals of 95% are shown. (D) Reaction specificity, as % of samples considered negatives by RT-qPCR that were also negative by RT-LAMP. Wilson’s binomial confidence intervals of 95% are shown.