Lateral flow biosensor combined with loop-mediated isothermal amplification for simple, rapid, sensitive, and reliable detection of Brucella spp.

Abstract

Brucella species is responsible for brucellosis in human and animals, which is still of public health, veterinarian, and economic concern in many regions of the world. Here, a novel molecular diagnosis assay, termed loop-mediated isothermal amplification coupled with nanoparticles-based lateral flow biosensor (LAMP-LFB), was developed and validated for simply, rapidly, and reliably detecting all Brucella spp. strains. A set of six primers was designed based on the Brucella-specific gene Bscp31. The Brucella-LAMP results were visually reported by biosensor within 2 mins. A variety of bacterial strains representing several Brucella species, as well as several Gram-negative and Gram-positive bacterial species were used to determine the analytical sensitivity and specificity of the assay. Optimal LAMP conditions were 63°C for 40 mins, and the assay’s sensitivity was found to be 100 fg of genomic DNA in the pure cultures. No cross-reactions to non-Brucella strains were obtained; thus, analytical specificity of LAMP-LFB assay is of 100%. Using the protocol, 20 mins for rapid DNA preparation followed by isothermal amplification (40 mins) combined with biosensor detection (2 mins) resulted in a total assay time of approximately 65 mins. In the case of 117 whole blood samples, 13 (11.11%) samples were Brucella-positive by LAMP-LFB, and the diagnostic accuracy was 100% when compared to the culture-biotechnical method. In conclusion, Brucella-LAMP-LFB technique developed in this study is a sensitive and specific method to rapidly identify all Brucella spp. strains, and can be applied as a potential diagnostic tool for brucellosis in basic, clinical, and field laboratories.