Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

Abstract

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human β-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

Fig 1. Screening primer performance at a low copy number of SARS-CoV-2 RNA.

(A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [34]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR^PC) or no template (TTR^NTC), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.

Fig 2. Evaluation of the optimized primer concentrations based on limit of detection and specificity.

(A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.

Fig 3. Effect of primer multiplexing and supplements.

(A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.

Fig 4. Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples.

(A) Correlation of Ct values with BGI RT-PCR kit vs. other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct < 37.0 and TTR ≤ 13.2’ respectively. (D) Distribution of human ACTB TTRs. (E) Representative fluorescent readouts and phenol red colour with RT-LAMP reactions at 60 minutes in (C and D) with the clinical NP samples. (F) ROC curve evaluating RT-LAMP performance with 21 positive and 21 negative NP samples from Ecuador. TTR ≤ 41’ was defined as the cut-off to distinguish positive from negative samples with 100% specificity and sensitivity. (G) Distribution of RT-LAMP TTRs vs. RT-PCR Ct values for gene E with samples in (F). RT-PCR and RT-LAMP positives were defined by Ct ≤ 30.0 for gene E and TTR ≤ 41’ respectively. (H) Distribution of human ACTB TTRs of samples in (F).

Fig 5. Direct RT-LAMP on raw clinical NP samples without RNA extraction.

(A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs. BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.

Fig 6. Direct RT-LAMP on raw clinical NP samples of Colombia and Ecuador.

(A) ROC curve evaluating the optimized direct RT-LAMP performance on 118 positive and 41 negative clinical NP samples from Colombia. (B) Distribution of the RT-LAMP TTRs vs. U-TOP Seasun RT-PCR Ct values for Orf1ab, and the sensitivity of RT-LAMP at the indicated Ct intervals with samples in (A). (C) Distribution of ACTB TTRs of samples in (A). (D) Plot of TTRs vs. Ct values in a simulation RT-LAMP test with randomly chosen Colombian samples, and the sensitivity at the indicated Ct intervals with these samples. (E) ROC curve analysis validating the simulation RT-LAMP test in (D). (F) Distribution of ACTB TTRs of samples in (D). (G) ROC curve analysis of the direct RT-LAMP with 21 positive and 21 negative NP samples of Ecuador. (H) Distribution of the direct RT-LAMP TTRs vs. RT-PCR Ct values for E gene, and the sensitivity at the indicated Ct values for samples in (G). (I) Distributions of ACTB TTRs of samples in (G). Dotted lines in distribution graphs indicate cutoffs.

Fig 7. Direct RT-LAMP with FluoroPLUM.

(A) FluoroPLUM readout of RT-LAMP assessment of the boxed wells in (B). Each solid line represents one reaction, monitored for 45 minutes, and quantified as ‘PLUM reading units’. N1A5 (red) and N1V5 (green) are examples of positive and negative samples, respectively. The red dash line is the average reading of all the wells in the plate (B) for the first 3 minutes. (B) Image of RT-LAMP reactions at the end of the experiment. (C) Slope_20-40 for the two reactions indicated in (A) and (B). (D) ROC curve evaluating FluoroPLUM performance using slope_20-40 values. (E) Distribution of slope_20-40 values vs. BGI RT-PCR Ct values. Dotted lines indicate cutoffs (RT-LAMP: Slope_20-40 > 0.0048 = positive, RT-PCR: Ct < 37 = positive). (F) Distribution of slope_20-40 for human ACTB in clinical NP samples. (G) Images of color changes for the 30 positive (red) and 29 negative (blue) clinical NP samples. Asterisks: Three samples with no amplification of ACTB, two of which showed amplification in a repeat run (bottom image). (H) Sensitivity with end-point data in (G).