Optimization of sample preparation and bacterial DNA enrichment procedures to improve performance of molecular detection of Brucella species and bacteria known as causative agents of cross-reactions in serological tests for brucellosis in tissue samples

Funding period: 2022–2024
Lead: Mingsong Kang
Total GRDI funding: $71,000

Brucellosis is a zoonotic disease that can lead to significant economic losses in the animal industry and pose severe threats to human health. Although it has now been effectively controlled in livestock in many countries, including Canada, wildlife reservoirs of brucellosis are still known to exist in Canada. In addition, this disease is still endemic in some developed countries and in most developing countries, which poses a potential threat to the Brucella-free status of Canada's livestock. Therefore, Canada's surveillance and import control programs are essential lines of defence against brucellosis outbreaks in the nation's livestock.

Molecular assays can be considered potential alternatives to conventional bacterial culture techniques for confirmatory testing of the Brucella organism to identify suspect or clinical cases. Moreover, molecular assays have also been evaluated as potential techniques for detecting Brucella DNA directly from tissue samples. Recently, PCR-based next-generation sequencing of the 16S rRNA gene has been evaluated to analyze the whole microbial profile in tissue samples to identify known or potential causative agents of several infectious diseases. Though these molecular techniques are well known for their simplicity, efficiency and robustness, they have not been widely applied because of several challenges related to sample preparation or DNA purification and enrichment processes.

The goal of this project is to improve and stabilize the performance of molecular tests for detecting Brucella DNA from tissue samples by optimizing procedures for sample preparation and bacterial DNA enrichment using several different biochemical methods combined with different commercial DNA purification kits. In addition, 16s rRNA gene amplicon sequencing will be evaluated to detect Brucella and other bacteria known to be causative agents of cross-reactions in brucellosis serological tests in tissue samples. If successful, this project will establish advanced methodologies for sample preparation and bacterial DNA enrichment, which could be broadly implemented to detect other pathogenic bacteria in similar tissue samples.