De Buck, JeroenMiao, Zhuohan2022-07-222022-07-222022-07-20Miao, Z. (2022). Development of a Gram-type Specific Method for Detection of Bovine Mastitis Pathogens by Combining Loop-mediated Isothermal Amplification (LAMP) and Split Trehalase Technologies (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/114870https://dx.doi.org/10.11575/PRISM/39931Bovine mastitis, which results mainly from intramammary infections (IMI) caused by Gram-positive or Gram-negative bacteria, is a huge burden on the global dairy industry. Oftentimes the presence of an infection can be deduced by measuring the somatic cell count (SCC) or released enzymes, or directly detected by the presence of mastitis pathogens. While many laboratory diagnostic methods (e.g., SCC determination, bacteria culturing, qPCR) exist, few have been modified and adopted for on-farm use due to the requirement of sterility, dedicated equipment and well-trained personnel for implementation, time restraints, and accuracy. As such, the development of new methods that can sensitively and specifically detect mastitis-causing pathogens on-farm would be very valuable. We aimed to detect and distinguish Gram-positive and Gram-negative mastitis bacteria to inform on the proper and prompter treatment/antibiotic to prescribe through two steps: (i) develop PCR and LAMP assay for a point-of-care (POC) amplification of Gram-positive and Gram-negative bacteria and (ii) use a modified amplicon binding split trehalase assay (ABSTA) to detect this amplification by a specific protein-DNA binding and the complementation of split trehalase. To do this, split trehalase fusion proteins HisTreA-C-SpoIIID and HisTreA-N-SpoIIID were purified, and the binding with modified oligonucleotides incorporated with SpoIII-specific recognition sequences was tested to verify and optimize the complementation conditions. Next, recognition sequences were introduced in Gram-type specific PCR primers, allowing for specific detection of PCR amplicons by ABSTA. We also integrated the recognition sequences in three Gram-type specific LAMP primer sets designed based on the pathogens’ 16S rRNA genes, allowing for specific detection of Staphylococcus, Streptococcus and Gram-negative bacteria LAMP products by ABSTA. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. Binding with purified PCR products demonstrated Gram-type detection specificity with the protein reagents, whereas the complementation appeared to be inhibited by agents in unpurified PCR products. The sensitivity of detection of bacterial genomic DNA of Escherichia coli, Staphylococcus devriesii and Streptococcus uberis ranged from 2 to 24. The sensitivity of detection of E. coli in spiked milk samples was 11 CFU/ml, but 4.9 × 104 CFU/ml and 2.0 × 105 CFU/ml for S. devriesei and S. uberis, respectively. The analytical specificity of the newly designed LAMP primer sets was evaluated with ten mastitis isolates at two genomic DNA copy number levels. In conclusion, the combination of bacterial genetic target amplification by LAMP and ABSTA demonstrated high sensitivity and specificity towards genomic DNA of Gram-positive and Gram-negative bacteria, enhancing the potential of developing a timesaving, user-friendly and cost-effective on-farm diagnostic method for educated treatment decisions of bovine mastitis causative pathogens.University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.BiologyMicrobiologyBiology--MolecularVeterinary Sciencemaster thesis