Nanoparticle-based biosensors coupled with loop-mediated isothermal amplification for rapid, visual, and in-field identification of pathogenic Leptospira

Leptospirosis is a worldwide re-emerging zoonotic infectious disease caused by pathogenic spirochetes of the Leptospira genus. The clinical symptoms at leptospirosis onset are similar to those of other febrile illnesses, and early etiological identification of pathogenic Leptospira spp. is crucial to initiate immediate treatment and limit transmission. Here, we report a new molecular diagnostic assay termed loop-mediated isothermal amplification combined with a visual gold nanoparticle-based lateral flow biosensor (LAMP-AuNP-LFB) for rapid, specific, sensitive, and visual identification of pathogenic Leptospira strains. The AuNP-based LFB used here was successfully manufactured according to our device manual. The successful design of our unique Leptospira-LAMP primers was based on the lipL41 gene from reference strains of 15 serovars representing 15 serogroups of pathogenic Leptospira spp. in China. The reaction conditions, including amplification temperature and time, were optimized, and the sensitivity and specificity of our assay were verified. The feasibility of our assay was tested through field mouse and leptospirosis clinical samples. The AuNPs-based LFB used our study was successfully manufactured. The unique pathogenic Leptospira degenerate primers were successfully designed based on the lipL41 gene. The optimal Leptospira-LAMP-AuNP-LFB detection process, including rapid nucleic acid isolation (less than 5 min), LAMP reaction (67 °C for 35 min), and visual AuNP-LFB readout (less than 2 min), could be accomplished within 45 min. Our assay detected target genes with as few as 5 copies per test and showed no cross-reactivity with other microbes in our study. The Leptospira-LAMP-AuNP-LFB assay results were consistent with culture-sequencing results in field mouse and leptospirosis clinical samples. The Leptospira-LAMP-AuNP-LFB assay is a valuable tool and has tremendous potential as a point-of-care (POC) testing approach for screening for pathogenic Leptospira infection, especially in resource-poor settings.