Visual and multiplex detection of nucleic acid sequence by multiple cross displacement amplification coupled with gold nanoparticle-based lateral flow biosensor

Abstract

Here, we reported on the establishment of a nearly instrument free, simple and rapid molecular technique, which incorporated multiple cross displacement amplification coupled with lateral flow biosensor (MCDA-LFB) for the visual, sensitive and specific detection of nucleic acid sequence. The MCDA-LFB assay was able to simultaneously detect and correctly differentiate multiple targets using a 40min amplification reaction followed by a 2min incubation of the products on the visualization biosensor. The biosensor was devised to detect three targets, a chromatography control, the MCDA target amplicon I and II, and the interpretation of test results is based on the appearance of red lines on the reaction pad. As a proof of concept, Shigella and Salmonella strains were detected by MCDA-LFB technique to demonstrate the availability of target analysis. The analytical sensitivity, specificity and practical application of MCDA-LFB assay were successfully evaluated in pure culture and blood samples, and the whole procedure, including specimen (blood sample) processing (35min), isothermal reaction (40min), and result reporting (5min), was completed within 80min. Therefore, the simplicity, rapidity and nearly apparatus-free platform of the MCDA-LFB assay make it practical for point-of-care testing, field detection, ‘on-site’ diagnosis and more.