Aims In this present study, the utility of a newly developed loop-mediated isothermal amplification (LAMP) and real-time PCR assays designed to amplify the virB gene region of Brucella melitensis was evaluated from human clinical specimens. Methods and Results Fifty-four culture-confirmed cases of brucellosis and 54 culture negative but clinically suspected cases of brucellosis were included in the study. Whole blood, serum and other nonblood specimens were collected and subjected to blood culture using automatic blood culture system, serological tests, LAMP assay and real-time PCR. Overall sensitivities of LAMP and real-time PCR assays were 67 center dot 5 and 68 center dot 3% respectively. For nonblood clinical specimens, we noticed a marked increase in the sensitivities of LAMP (88 center dot 9%) and real-time PCR (100%) assays. Conclusions Performance of LAMP and real-time PCR was not satisfactory for whole-blood specimens because of the low abundance of bacteria or DNA. On the other hand, using nonblood specimens, both the assays showed higher sensitivity and specificity which makes them a good alternative for the rapid diagnosis of human brucellosis. Significance and Impact of the Study The developed LAMP and real-time PCR assays are a specific and rapid diagnostic tool for direct and early detection of Brucella in clinical specimens.