Molecular detection of brucella in goat blood samples: Comparative analysis of three DNA extraction methods using brucella vaccine as a control

Abstract

Brucellosis, a zoonotic disease caused by Brucella species, poses a significant threat to both livestock and human populations. In regions with a history of abortion in goat herds, the molecular detection of Brucella becomes crucial for disease management and prevention. This study aimed to assess the efficiency of three different DNA extraction methods, including a commercially available kit, Proteinase K method (PKM), and non-enzymatic salting-out method, in extracting Brucella DNA from goat blood samples.Fifteen goat blood samples and two Brucella vaccine samples were subjected to DNA extraction using these methods. The DNA extracted from goat blood samples using the commercial kit exhibited a mean concentration of 87.4 ± 9.35 ng/µl, while PKM yielded a concentration of 65.15 ± 21.87 ng/µl, and the non-enzymatic salting-out method resulted in 32.71 ± 24.55 ng/µl. The 260/280 ratios ranged from 0.75 to 1.07, indicating varying degrees of DNA purity. DNA extracted from the vaccine samples showed a mean concentration of 28.2 ± 2.54 ng/µl (commercial kit), 14.5 ± 3.53 ng/µl (PKM), and 8.05 ± 2.75 ng/µl (non-enzymatic salting-out method) with corresponding 260/280 ratios. PCR amplification targeting the bcsp gene revealed notable differences in Brucella detection. Among blood samples, only three samples extracted using the PKM method exhibited positive bands, while both vaccine DNA samples consistently produced bands. Likewise, 1 isolated sample from the Commercial Kit extraction displayed a positive band, and both vaccine DNA samples showed bands as well. Conversely, none of the DNA samples extracted using the non-enzymatic salting-out method yielded positive bands for the bcsp gene. Gel electrophoresis confirmed the presence of well-defined bands, indicating the integrity of DNA. These findings suggest that the choice of DNA extraction method can significantly impact the detection of Brucella in goat herds. The Proteinase K method appeared to be the most effective among the methods tested. This study provides valuable insights for the accurate diagnosis of Brucella in goat populations with a history of abortion in Rajnandgaon District, Chhattisgarh, facilitating timely intervention and disease control measures.