Lateral flow biosensor based on LAMP-CRISPR/Cas12a for sensitive and visualized detection of Salmonella spp.

Abstract

Salmonella is a major pathogen that causes serious foodborne diseases in humans and poses a serious threat to food safety and public health worldwide. Its rapid and accurate diagnosis is essential to prevent bacterial contamination of food. This study aimed to develop a lateral flow biosensor (LFB) based on loop-mediated isothermal amplification (LAMP)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 12a (Cas12a) that is affordable and can aid in visually detecting Salmonella in a food sample. False positives were prevented by synthesizing CRISPR ribonucleic acid (crRNA) with a selected 21-base pair protospacer that did not overlap with the used LAMP primers. A single-stranded deoxyribonucleic acid (DNA) probe labeled with biotin and 6-Carboxyfluorescein (6-FAM) was used for designing the LFB. The duration of these processes was as follows: DNA extraction (20 min), LAMP (60 min), Cas12a cleavage (5 min), and LFB (5 min). The detection limit was 1.22 × 10° CFU/mL in pure culture. The validity of the LAMP-CRISPR/Cas12a based LFB was verified by detecting Salmonella in onions, melon, and salami. Consequently, the developed LAMP-CRISPR/Cas12a based LFB assay show the potential for rapid on-site detection of Salmonella with high specificity, sensitivity, convenience, mobility, low cost, and affordability. • LFB based on LAMP-CRISPR/Cas12a was developed for detection of Salmonella. • The developed method demonstrated suitability for naked-eye detection of Salmonella. • The detection can be completed within 100 min for on-site detection.