Research ArticleCORONAVIRUS

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

See allHide authors and affiliations

Science Translational Medicine  12 Aug 2020:
Vol. 12, Issue 556, eabc7075
DOI: 10.1126/scitranslmed.abc7075
  • Fig. 1 Sensitivity of the RT-LAMP assay determined using IVT RNA.

    (A) Defined numbers of in vitro transcribed (IVT) RNA molecules of the SARS-CoV-2 N gene were added to the RT-LAMP reaction and incubated at 65°C. At indicated times, samples were removed from the heating block and cooled on ice to stop the reaction. Photographs were taken using the color scanner function of an office copy machine and show the red to yellow color change in positive samples. (B) The RT-LAMP reaction product (2.5 μl) was analyzed on a 2% agarose gel. The typical band pattern of a successful RT-LAMP reaction was visible in the samples with 100 or more SARS-CoV-2 RNA molecules, i.e., in those samples that showed a color change from red to yellow after 30 min.

  • Fig. 2 Sensitivity and specificity of the RT-LAMP assay compared to RT-qPCR using clinical samples.

    RNA samples isolated from 95 pharyngeal swab specimens were analyzed by the RT-LAMP assay using a 96-well plate. The RT-LAMP reaction was incubated at 65°C, and the incubation was interrupted at different time points by cooling on ice for 30 s. (A) Photograph of the 96-well plate after a 30-min incubation at 65°C, taken with a mobile phone. Wells with a yellow color indicate successful RT-LAMP amplification of a fragment of the SARS-CoV-2 N gene (using the N-A primer set). (B) Quantification of the red-to-yellow color change in all wells using spectrophotometric OD measurements. The color value at the given time points is quantified as the difference between the wavelengths of the two absorbance maxima of phenol red: ΔOD = OD434 nm – OD560 nm. Yellow (positive) samples yield a ΔOD of about 0.3 to 0.4. Each line represents one sample. For each sample, the line color indicates the CT (cycle threshold) value obtained from RT-qPCR data (using the E-Sarbeco primers) (15). (C) Scatter plot of ΔOD values at the 30-min time point from (B) compared to CT values from RT-qPCR. Each dot is one sample (well).

  • Fig. 3 Detection of SARS-CoV-2 RNA using the RT-LAMP assay.

    (A) Scatter plot shows a comparison of RT-LAMP assay results and RT-qPCR results for RNA samples tested on 10 96-well plates. The RNA extraction method (QC, QiaCube, a column-based method; QS, QiaSymphony, a bead-based method) is indicated. The time point for measurement by the colorimetric RT-LAMP assay was 30 min after the start of the 65°C incubation. The 96-well plate shown in Fig. 2 is not included here. Table 1 shows numbers of samples stratified according to the results of the RT-LAMP and the RT-qPCR assays. (B) Sensitivity (right) and specificity (left) of the RT-LAMP assay [derived from data in (A) and Table 1] are shown. The specificity is the fraction of RT-qPCR–negative samples correctly identified as negative by the RT-LAMP assay. For sensitivity, the RT-qPCR–positive samples were stratified by CT values into three bins (as indicated by x axis labels), and for each bin, the sensitivity is given as the fraction of qPCR-positive samples in the respective CT bin that have also given a positive result in the RT-LAMP assay. The thick black lines indicate the values of these fractions (i.e., the specificity and sensitivity estimates); the black boxes indicate the corresponding 95% confidence intervals (Wilson’s binomial confidence interval). (See also table S2).

  • Fig. 4 Multiplexed sequencing of RT-LAMP reaction products (LAMP-sequencing).

    (A) Workflow for LAMP-sequencing is shown. A plate of 96 barcoded (BC) adapters with unique molecular identifiers (UMIs) and mosaic ends (ME) was used as a seed plate for Tn5 tagmentation of all RT-LAMP reaction products. After tagmentation, each plate was pooled individually, followed by removal of excess adapters using size selection. Each pool of tagmentation products was then amplified using primers with plate-specific barcodes, and the PCR products were analyzed by Illumina sequencing. (B) Comparison of the outcome of the three assays: LAMP-sequencing (purple, negative; green, positive; gray, too few UMIs), RT-LAMP (after 30-min incubation, y axis), and RT-qPCR (x axis). Each dot represents one sample. If a substantial number of the sequencing reads contained SARS-CoV-2 RNA, the sample was called positive (green), if not, then it was called negative (purple). For some samples (gray), no LAMP-sequencing call could be made due to too few UMIs. (See also Table 2). (C) Although the RT-LAMP assay was scored after a 30-min incubation at 65°C (left), LAMP-sequencing was performed only after the samples had been incubated for another 10 min (15 min for one plate). This panel shows the RT-LAMP assay outcome (y axis) scored after the full incubation time, whereas the RT-qPCR CT values (x axis) and LAMP-sequencing results are the same as in (B).

  • Fig. 5 Swab–to–RT-LAMP assay of clinical pharyngeal swab samples.

    (A) Skipping a prior RNA isolation step, pharyngeal swab samples were subjected to the RT-LAMP assay either directly (left) or after 5 min of heat treatment at 95°C (right). For each sample, scatter plots are used to compare the swab–to–RT-LAMP assay results (ΔOD values) with the results of RT-qPCR (CT values). The measurement time point was 30 min after the start of the 65°C incubation. (B) Shown is the sensitivity (right) and specificity (left) of the swab–to–RT-LAMP assay [derived from the data in (A)] using the decision threshold indicated by the horizontal gray line in (A). Specificity and sensitivity values (thick lines) are shown with their 95% confidence intervals (boxes) as in Fig. 3, with blue indicating the direct swab–to–RT-LAMP assay and red indicating the hot swab–to–RT-LAMP assay. (Also see table S3).

  • Fig. 6 Colorimetric readouts of the swab–to–RT-LAMP assay over time.

    (A) The colorimetric readouts (ΔOD) for the direct (left) and hot (right) swab–to–RT-LAMP assays were assessed every 10 min. Heterogeneity is notable at the early time points. ΔOD values at the zero time point were not measured for the hot swab–to–RT-LAMP assay. Also, the 40-min time point was not available for one plate. The kink in some lines at 30 min (right) was due to a transient equipment malfunction. (B) Comparison of two scoring schemes. The readout used in Fig. 5 to score the direct (left) and hot (right) swab–to–RT-LAMP assays, namely, ΔOD at 30 min, is shown on the y axis, and compared to an alternative score, namely, the difference between the ΔOD signals at 30 min and at 10 min after the start of incubation, shown on the x axis. The latter shows better separation between positive and negative samples.

  • Table 1 Shown is RT-qPCR and RT-LAMP testing of 768 clinical samples stratified into CT value bins (see Fig. 3A).

    Fig. 3B and table S2 show specificity and sensitivity values calculated from these numbers.

    RT-LAMP
    CTPosNegSum
    RT-qPCRPos0–2551051
    25–3028230
    30–3541620
    35–4001616
    NegNeg2649651
    Sum85683768
  • Table 2 Summary of LAMP-sequencing results.

    The cross tabulation of RT-qPCR and RT-LAMP assay results shown in Table 1 have been split into samples where sequencing of RT-LAMP reaction products (LAMP-sequencing) was positive (Pos), negative (Neg), or inconclusive (too few reads) (see also Fig. 4).

    RT-LAMP
    CTPosNegSum
    LAMP-
    sequencing
    PosRT-qPCRPos0–2549049
    25–3028028
    30–35404
    35–40000
    NegNeg000
    NegRT-qPCRPos0–25000
    25–30022
    30–3501616
    35–4001616
    NegNeg2637639
    Too few readsRT-qPCRPos0–25202
    25–30000
    30–35000
    35–40000
    NegNeg01212
    Sum85683768
  • Table 3 Shown is RT-qPCR and RT-LAMP testing of 592 clinical samples stratified into CT value bins (see Fig. 5A).

    Fig. 5A and table S3 show specificity and sensitivity values calculated from these numbers.

    Hot swab–to–RT-LAMPRT-LAMP
    CTPosNegSum
    RT-qPCRPos0–2538442
    25–3017522
    30–3552328
    35–4003636
    NegNeg1214215
    Sum61282343
    Direct swab–to–RT-LAMPRT-LAMP
    CTPosNegSum
    RT-qPCRPos0–2515116
    25–3061117
    30–3522123
    35–4032326
    NegNeg9144153
    Sum35200235

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/556/eabc7075/DC1

    Materials and Methods

    Fig. S1. Design of the study.

    Fig. S2. Comparison of the RT-LAMP assay with CT values from RT-qPCR using primer sets E-Sarbeco and N-Sarbeco.

    Fig. S3. Comparison of the RT-LAMP assay using primer set 1a-A with CT values from RT-qPCR using primer set E-Sarbeco.

    Fig. S4. Analysis of LAMP-sequencing reads.

    Fig. S5. Sample classification with LAMP-sequencing.

    Fig. S6. RNA stability and detection limit of the RT-LAMP assay in pharyngeal swab specimens with IVT RNA.

    Fig. S7. Swab–to–RT-LAMP assay titration of positive COVID-19 specimens.

    Table S1. Sequences of primers and amplicons used in this study.

    Table S2. Sensitivity and specificity of the RT-LAMP test from Fig. 3B.

    Table S3. Sensitivity and specificity of the RT-LAMP test from Fig. 5B.

    Table S4. Primers used for LAMP-sequencing.

    Table S5. Overview of time requirements for various sample handling steps.

    Data File S1. Raw data for figs. S6 and S7.

  • The PDF file includes:

    • Materials and Methods
    • Figure S1. Design of the study
    • Figure S2: Comparison of the RT-LAMP assay with CT values from RT-qPCR using primer sets E-Sarbeco and and N-Sarbeco
    • Figure S3: Comparison of the RT-LAMP assay using primer set 1a-A with CT values from RT-qPCR using primer set E-Sarbeco
    • Figure S4: Analysis of LAMP-sequencing reads
    • Figure S5: Sample classification with LAMP-sequencing
    • Figure S6: RNA stability and detection limit of the RT-LAMP assay in pharyngeal swab specimens with IVT RNA
    • Figure S7: Swab-to-RT-LAMP assay titration of positive COVID-19 specimens
    • Table S1: Sequences of primers and amplicons used in this study
    • Table S2: Sensitivity and specificity of the RT-LAMP test from Figure 3B
    • Table S3: Sensitivity and specificity of the RT-LAMP test from Figure 5B
    • Table S5: Overview of time requirements for various sample handling steps

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article