Rapid and Specific Detection of B. melitensis Targeting BMEI1661 Gene Using Loop-mediated Isothermal Amplification (LAMP) Combined With Lateral Flow immunoassay (LFIA).

B. melitensis is the most pathogenic zoonotic species of Brucella transmitted to animals through fetal secretions, placenta, and vaginal discharges of infected animals and humans by ingesting unpasteurized milk, dairy products, and raw meat. Early detection of B. melitensis is essential for timely intervention and control of the disease. The gold standard diagnostic methods, such as culture, are time-consuming and may take several weeks aiding to the disease spread. Loop-mediated isothermal amplification assay (LAMP) is widely used to detect infectious pathogens. LAMP can be utilized as a rapid point-of-care test, but has lower specificity which can be enhanced by combining this test with lateral flow immunoassay. No point-of-care test is available for detecting Brucella melitensis in clinical samples. Herein, we developed a LAMP coupled with lateral flow immunoassay (LFIA) for the specific detection of B. melitensis. The sensitivity of LAMP-LFIA was found to be 12.1 fg of genomic DNA isolated from the organism, which is 100-fold more sensitive to conventional PCR and equally sensitive to Real-time (RT-PCR). Moreover, the assay demonstrated high specificity when tested against other Brucella and non-Brucella species. The infective dose of B. melitensis is relatively low for humans, which may remain undetected by conventional PCR, but will be detected using the new technique.