title: Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.
abstract: Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243122 India.
authors: Bikash R
authors: BR
authors: Prusty
authors: Division of Bacteriology, Indian Veterinary Research Institute, Izatnagar, India.
authors: Pallab
authors: P
authors: Chaudhuri
authors: Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, India.
authors: V K
authors: VK
authors: Chaturvedi
authors: Center for Wildlife, Indian Veterinary Research Institute, Izatnagar, India.
authors: Mohini
authors: M
authors: Saini
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243122 India.
authors: B P
authors: BP
authors: Mishra
authors: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243122 India.
authors: Praveen K
authors: PK
authors: Gupta
keywords: Bovine brucellosis; Bovine semen; Loop-mediated isothermal amplification assay; bcsp31; omp2a