A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settings

Abstract

Background: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR.

Methods: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay.

Results: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site.

Conclusion: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.

Keywords: Extraction-free LAMP; Resource-limited settings; SARS-CoV-2; qRT-PCR.

Fig. 1

A Comparison of different primers on 5 positive and 2 negative qRT-PCR samples (primers taken from different publications as indicated) after 40 min, neg—H₂O control; B Temperature gradient to test LAMP stability, results were recorded after 30 min, primers are from the indicated references; C Determination of LAMP sensitivity on a tenfold serial dilution of two samples with known copy numbers (sample #1: 5540 copies/µl, sample #2: 4200 copies/µl)

Fig. 2

Sensitivity and specificity of LAMP assay compared to qRT-PCR on samples from St. George’s Hospital, London, the crosstables show true positives (pos/pos), true negatives (neg/neg), false positives (neg/pos) and false negatives (pos/neg) of the LAMP assays compared to qRT-PCR; A N primers and B Orf1ab primers from Yu et al. [16]

Fig. 3

LAMP of non-extracted swab samples from GOSH, London (30 min incubation); A N-LAMP and Orf-LAMP results for 12 non-extracted patient samples compared to qRT-PCR, pos—positive results, neg—negative result; B N-LAMP assay to test the effect of VTM buffer (conditions A, B, C, D) and heat-inactivation (conditions C, D) on SARS-CoV-2 positive samples (condition B, D, E); + and − indicate presence or absence of the according conditions

Fig. 4

Results for N-LAMP testing of non-extracted swab samples at KUHeS, Malawi, the crosstables show true positives (pos/pos), true negatives (neg/neg), false positives (neg/pos) and false negatives (pos/neg) of the LAMP assays compared to qRT-PCR