A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Abstract

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

Figure 1

Limit of detection of the two different RT-LAMP formats and of RT-PCR. (A) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. (B) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. (C) Limit of detection of ten replicates of the two test formats. (D) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1).

Figure 2

Detection of SARS-CoV-2 in NP samples using RT-LAMP. (A) Comparison of RT-LAMP and RT-PCR results. The Ct values (RT-PCR results) of 126 COVID-19 positive patients (y-axis) were compared to the RT-LAMP readout (x-axis) taken after 30 min of incubation at 65 °C (positive, +/yellow; negative, −/pink). The dotted red line indicates the Ct below, which there is 100% agreement between RT-LAMP and RT-PCR. (B) Sensitivity of the RT-LAMP assay across different ranges of Ct values (which reflect different viral loads). The thicker horizontal lines indicate the specificity calculated for the indicated Ct range (according to the data of panel (A) and Table 1). The vertical lines indicate the corresponding 95% confidence intervals.

Figure 3

Limit of detection of the saliva RT-LAMP assay. Spike-in experiments of a healthy donor saliva with (A) tenfold dilutions of in vitro transcribed (IVT) viral RNA (N-gene) and (B) in vitro propagated SARS-CoV-2 virions. Saliva samples were processed as described in “Materials and methods” and 2 μL were analyzed by colorimetric RT-LAMP. PFUs plaque forming units. Figures are representative of three independent experiments.

Figure 4

Detection of SARS-CoV-2 in saliva samples using RT-LAMP. RT-LAMP analysis of saliva samples of confirmed COVID-19 patients who (A) induced or not (B) salivation before sample collection. (C) Comparison of RT-LAMP and RT-PCR results. The Ct values (RT-PCR results) of 39 COVID-19 positive patients (y-axis) were compared to the RT-LAMP readout of the matched saliva samples (x-axis), after 30 min of incubation at 65 °C (positive, +/yellow; negative, −/pink). Black circles—direct saliva; orange circles—RNA extracted from saliva. (D) RNAs from the saliva of false negative samples (as determined by the direct saliva test) were extracted using plasmid DNA miniprep columns (ZR Plasmid Miniprep-Classic Kit, Zymo Research) and re-analyzed. NTC no template control. Cts (N gene) obtained for the paired NP samples are indicated.

Figure 5

Analytical sensitivity of an in-house-made colorimetric RT-LAMP assay. Tenfold dilutions of in vitro transcribed (IVT) viral RNA (N-gene) were amplified via RT-LAMP and detected using a colorimetric buffer together with (A) RTx (New England Biolabs) and Bst LF (homemade), (B) MashUP RT (homemade) and Bst 2.0 (New England Biolabs) or (C) MashUP RT (homemade) and Bst LF (homemade). The in-house-made setup was next used to detect SARS-CoV-2 sequences in (D) RNAs extracted from the NP fluid (NPF) and (E) saliva samples of COVID-19 positive patients. The reactions were incubated at 65 °C for 30 min. In (D) and (E) the Cts (N gene) obtained for the paired NP samples are indicated. NTC no template control, HD healthy donor. Figures are representative of three independent experiments.

Figure 6

Alternative colorimetric detection based on the complex murexide-zinc. (A) A strong color change from yellow to pink is observed when pyrophosphate (PPi) is added to a solution containing Zn-MX. Tenfold dilutions of in vitro transcribed (IVT) viral IVT RNA (Ngene) were amplified via RT-LAMP and detected using phenol red (B) or Zn-MX (C). Amplification was confirmed by agarose gel electrophoresis (AGE). Saliva samples of a healthy donor (HD) and of nine COVID-19 patients were analyzed by RT-LAMP followed by detection with Zn-MX (D) or phenol red (E) and amplification was confirmed by AGE (Original gel images in Fig. S1).