Development and application of the novel visual loop-mediated isothermal amplification of omp25 sequence for rapid detection of Brucella sp.

Abstract

A novel visual Loop-mediated isothermal Amplification (LAMP) method for detecting Brucella sp. was established. LAMP primers were designed by targeting the species-specific omp25 gene. In this LAMP reaction, manganous ion and calcein were added which allows the results to be simply judged by naked eye under daylight by color change from orange to green in a closed-tube system without expensive equipment and it took <60 min at 63°C. The assay correctly identified 3 Brucella sp. strains but did not detect 8 other bacterial species. Detection limit of genomic DNA of Brucella sp. was 10 pg. The assay was also successfully applied for the detection ofBrucella sp. from contaminated milk (as low as 1.3×l03CFU mL-1) and infected mice tissues. The assay shows enormous potential for rapidly, expediently and accurately detecting Brucella sp. from food and clinical samples. © Medwell Journals, 2011.