Development of a labelled-LFIA coupled with LAMP for the rapid and specific detection of B. melitensis targeting BMEI1661 gene

Abstract

Abstract The primary cause of Brucellosis in sheep, goats, human and other animal species is B. melitensis. Despite being widely acknowledged as the gold standard method, the isolation and identification of B. melitensis cannot currently meet the criteria for early diagnostic strategies. Conventional PCR methods and immunological assays can detect B. melitensis, but their use in basic laboratories is constrained by the need for sophisticated instruments. A prompt and accurate diagnosis is essential to prevent the spread of infection to human and animals. The aim of this study was to set up a rapid and specific point of care diagnostic test for the detection of B. melitensis, which could be used routinely. Hence, a Loop mediated isothermal amplification (LAMP) coupled with lateral flow immunoassay (LFIA) was optimized for the specific detection of B. melitensis. The LAMP primers were tagged to generate product labelled with digoxigenin and biotin and the labelled LAMP amplicons were detected using LFIA with streptavidin-gold nanoparticle as a capture reagent. The assembly of gold nanoparticle at the test and control line yielded a characteristic red colour band. The sensitivity of B. melitensis LAMP-LFIA assay was 12.1fg. The assay was 100 fold more sensitive than conventional PCR and was in accordance with RT-PCR. The assay did not exhibit cross reactivity with the non-Brucella pathogens examined in this study or other Brucella species. The assay can be performed in a water bath at 65°C within 60 min and can detect upto12.1fg of genomic DNA isolated from the organism and 102 CFU/ml of B. melitensis in the spiked sample. Thus there is a great deal of potential for this assay to be utilised as a rapid field test for screening different secretions and excretions in suspected animals or their products which can tremendously reduce chances of disease transmission to both animal and human.