Loop-Mediated Isothermal Amplification Coupled Lateral Flow Assay to Detect Nucleic Acids in Clinical Samples for Brucella Species at Point of Care and Resource-Limited
Keywords:
Brucella species, IS711 gene, Labeled primers, LAMP-LFA, Resource-limited areas.Abstract
Brucellosis is a most common and neglected zoonotic disease caused by Brucella species. Brucellosis is a major public health challenge as both animals and humans are affected. Most of the Brucella diagnostic tools are focused around serological assays though these assays cannot detect active pathogenesis. Loop-mediated isothermal amplification (LAMP) coupled with lateral flow assay (LFA) was developed in the present study to detect IS711 gene of Brucella species in the clinical samples. Primers were designed and the inner primers were labeled with fluorescein isothiocyanate (FITC) and biotin at 5′ end. The double labeled amplicons were detected rapidly at the test line on an LFA specifically designed to detect full length specific amplicons within 5 minutes. The novel assay could detect Brucella species genomic DNA as little as 5 pg. Furthermore, the assay showed a significant specificity since no cross-reaction observed with genomic DNAs other bacteria species. In the clinical sample analysis, the novel assay sensitivity was 98.3%, highly reproducible and specificity was 100% when compared to the standard culture assay. To our knowledge, this is the first LAMP-LFA assay for detection of Brucella species. Based on the sensitivity, specificity, rapidity and convenience LAMP-LFA could be useful for laboratory testing and epidemiological surveys for brucellosis at resource-limited areas.
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