Development and clinical application of loop-mediated isothermal amplification combined with lateral flow assay for rapid diagnosis of SARS-CoV-2

Abstract

Background: The diagnostic assay leveraging multiple reverse transcription loop-mediated isothermal amplification (RT-LAMP) could meet the requirements for rapid nucleic acid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Methods: The devised assay merged the lateral flow assay with the RT-LAMP technology and designed specific primers for the simultaneous detection of the target and human-derived internal reference genes within a single reaction. An inquiry into the assay's limit of detection (LOD), sensitivity, and specificity was carried out. The effectiveness of this assay was validated using 498 clinical specimens.

Results: This LOD of the assay was determined to be 500 copies/mL, and there was no observed cross-reaction with other respiratory pathogens. The detection results derived from clinical specimens showed substantial concordance with those from real-time reverse transcription-polymerase chain reaction (RT-qPCR) (Cohen's kappa, 0.876; 95% CI: 0.833-0.919; p<0.005). The diagnostic sensitivity and specificity were 87.1% and 100%, respectively.

Conclusion: The RT-LAMP assay, paired with a straightforward and disposable lateral immunochromatographic strip, achieves visual detection of dual targets for SARS-CoV-2 immediatly. Moreover, the entire procedure abstains from nucleic acids extraction. The samples are lysed at room temperature and subsequently proceed directly to the RT-LAMP reaction, which can be executed within 30 minutes at a constant temperature of 60-65°C. Then, the RT-LAMP amplification products are visualized using colloidal gold test strips.

Trial registration: This study was registered at the Chinese Clinical Trial Registry (Registration number: ChiCTR2200060495, Date of registration 2022-06-03).

Keywords: COVID-19; Diagnostics; Isothermal amplification; Molecular testing; RT-LAMP; SARS-CoV-2.

Fig. 1

The plane structure diagram of the lateral flow strip. The lateral flow strip includes a sample pad (the zone used to load the LAMP product), a detection zone consisting of test line 1, test line 2, and control line, respectively immobilized with anti-Digoxin, anti- FITC, and biotin, as well as an absorbent pad. Flow through indicates the flow direction of the sample on the strip. The MAX lines indicate the position where the sample is loaded on the sample pad

Fig. 2

The RT-LAMP results of single target primer. The fluorescence amplification curves were shown on the top, and the coloration reaction results were shown on the bottom. N-1, novel coronavirus N gene primer 1; N-2, novel coronavirus N gene primer 2; ACTB-1 (A-1), internal reference gene human-derived ACTB gene primer 1; ACTB-2 (A-2), internal reference gene human-derived ACTB gene primer 2. NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 3

The RT-LAMP results of dual target primer. The fluorescence amplification curves were shown on the top, and the coloration reaction results were shown on the bottom. G1, N-1 and ACTB-1; G2, N-2 and ACTB-1; G3, N-1 and ACTB-2; G4, N-2 and ACTB-2. NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 4

The RT-LAMP results of lysate samples and nucleic acid samples. The fluorescence amplification curves were shown on the top, and the coloration reaction results were shown on the bottom. NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 5

The RT-LAMP results from different lysis times. The fluorescence amplification curves were shown on the top, and the coloration reaction results were shown on the bottom. NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 6

The results of RT-LAMP at different reaction temperatures. The fluorescence amplification curves were displayed on the top, and the coloration reaction results were shown on the bottom. NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 7

The coloration reaction results of RT-LAMP experiment with different reaction times. NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 8

The level of detection (LOD) obtained using RT-LAMP assays. A The coloration reaction results of RT-LAMP assays for pseudovirus samples at different concentrations (50, 100, 250, 500, 750, and 1000 copies/mL); B The coloration reaction results of 20 repeated RT-LAMP assays for 500 copies/mL pseudovirus samples. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line

Fig. 9

The coloration reaction results of RT-LAMP specificity test. FluA, influenza A virus; FluB, influenza B virus; HCoV-229E, human coronavirus 229E; HPIV, human parainfluenza virus; HRV, human rhinovirus; HmPV, human metapneumovirus; EV, enterovirus; HCMV, human cytomegalovirus; PC, positive control; NTC, negative control. T1 line, Test line 1 (ACTB gene); T1 line, Test line 2 (N gene); C line, Control line