A probe-corrected loop-mediated isothermal amplification–lateral flow assay platform for highly specific, point-of-care detection of Brucella abortus DNA

Abstract

Brucellosis, primarily caused by Brucella abortus, is a significant zoonotic disease affecting cattle worldwide. Accurate and early detection of B. abortus is crucial for effective disease control; however, existing diagnostic methods are limited by low sensitivity, lengthy processing times, and impracticality for field use. In this study, we developed a novel loop-mediated isothermal amplification–lateral flow assay (LAMP–LFA) platform that integrates a biotinylated DNA probe and fluorescein isothiocyanate–labeled primers to detect BruAb2_0168 of B. abortus. Unlike conventional LAMP-based diagnostics, our platform does not entirely prevent nonspecific amplification. Instead, it distinguishes target-specific products from nonspecific products by selective probe hybridization. Only true-positive amplicons hybridize with the probe to produce visible signals on the LFA strip, effectively eliminating the false-positive interpretations that typically limit the diagnostic reliability of LAMP-based methods. We optimized the amplification conditions (temperature, time, and primer/probe concentration) and LFA strip design to ensure high performance. Our platform achieved a detection limit of ≥8.85 CFU/mL, and no cross-reactivity was observed with other common pathogens. Validation using 20 whole-blood samples demonstrated that its diagnostic performance was statistically equivalent to that of polymerase chain reaction (area under the curve = 1.0, McNemar's p > 0.05). The assay reports results within 1 h, requires no instrumentation, and is suitable for use in field or resource-limited settings. Overall, our probe-corrected LAMP–LFA platform offers enhanced specificity, operational simplicity, and field-deployable utility, making it a valuable alternative for rapid and accurate B. abortus diagnosis. © 2024