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).