Purpose: The purpose of the present work was to establish a laboratory Loop-mediated Isothermal Amplification method for rapid detection of Brucella spp.
Methods: The Loop-mediated Isothermal Amplification(LAMP) method employs four primers which specifically recognize six distinct sequences of the target DNA and a bst DNA polymerase which has strand displacement activity. The specific LAMP primers of Brucella were designed according to a published sequence of gene omp 25 using the LAMP primer design support software program. The reaction was carried out at 65°C for 1 h and the products were examined by SYBR Green I stain or by 2.0% agarose gel electrophoresis. The reaction conditions including primers, temperature, time, bst polymerase concentration, dNTPs concentration were optimized. The LAMP reaction was carried out in a 25 μL total reaction mixture volume containing 1.6 μmol/L each of inner primers FIP and BIP, 0.2 μmol/L each of outer primers F3 and B3, 1.6 mmol/L dNTPs, 12.5 mmol/L KCl, 12.5 mmol/L (NH4)2SO4, 8.0 mmol/L MgSO4, 0.125% Triton X-100, 0.5 μL of bst DNA polymerase, and 2.0 μL of target DNA. The mixture was incubated at 65°C for 60 min in a heating block and then heated at 80°C for 10 min to terminate the reaction.
Results: The specificity of LAMP method with bsp primers were evaluated by blind amplification of DNA from 1 isolate of Brucella and 29 isolates of non-Brucella Gram-negative bacteria. Positive reactions were only observed on the Brucella isolate but no other bacteria. The sensitivity of the LAMP method was evaluated by running the reaction on serial diluted bacteria and determined to be 3.81×101CFU/mL.
Significance: The LAMP method developed in this work was specific and sensitive and can be potentially applied for rapid detection of Brucella spp. in food and environmental matrix.