A Closed Tube Loop-Mediated Isothermal Amplification Assay for Identification of BrucellaSpecies in Bull Semen

A closed tube loop-mediated isothermal amplification (LAMP) assay targeting omp25gene was performed for detection of Brucellasp in semen from infected bulls. The assay was achieved through the use of reaction master-mix for performing LAMP reactions. The reaction master-mix contained all reaction components at their optimized concentration including BstDNA polymerase and hydroxynaphthol blue (HNB) dye. The use of master-mix avoided opening of tubes after initial denaturation step required for adding BstDNA polymerase. The HNB dye allowed visual monitoring of positive and negative LAMP reactions through naked eyes and hence circumvented need of opening reaction tubes for electrophoretic analysis of LAMP products. The use of master-mix simultaneously minimized contamination of laboratories, saved time and simplified reaction set up. The assay was performed in a simple water bath making it cost-effective. It was found specific to Brucellaspp. only and could detect as few as 10 Brucellacolony forming units in experimentally contaminated semen and 10 fg DNA (nearly 3 genome copies of Brucella) in pure culture. This sensitivity was found equivalent to that of TaqMan probe- based real-time PCR and 10 times higher than conventional PCR. Overall, the optimized LAMP assay was rapid, simple in operation, result oriented, highly sensitive and inexpensive. This could be used as a diagnostic test for detection of Brucellain semen at breeding bull stations in developing countries where laboratories are generally poorly equipped.