A novel loop-mediated isothermal amplification assay coupled with lateral flow assay for differential detection of Brucella spp from Brucella abortus S19 vaccine strain.

Abstract

A Loop-Mediated Isothermal Amplification (LAMP) assay coupled with Nucleic-acid Lateral Flow Immunoassay (NALFIA) was developed to differentiate B. abortus S19, the vaccine strain, from other Brucella Species, including wild-type B. abortus. LAMP assay was carried out by targeting the eryC gene, which is absent in B. abortus S19. Specific ligands on the test and control lines of the LFA strip were spotted. The presence of a colored band only on the control line indicated the presence of B. abortus S19. In contrast, a colored line on both test and control lines indicated the presence of other Brucella Species. The assay was found to be very specific, as it did not cross-react with any other bacterial genera. The assay was found to be very sensitive, and the limit of detection was found to be 1.76 pg of genomic DNA of bacteria. In spiked milk samples, the assay could detect 2.89 × 10CFU of bacteria. Thus, the assay, which has discriminatory power in the detection of B. abortus S19 and other Brucella Species, can be used as nucleic acid-based DIVA in clinical samples of suspected cases of bovine brucellosis, which can be a potential source of transmission in both humans and animals.