| dc.description.abstract | Canine monocytic ehrlichiosis (CME), caused by the obligate intracellular bacterium
Ehrlichia canis, poses a significant threat to dogs globally. Early and accurate detection
of CME is critical for effective disease management and prevention. This thesis focuses
on the development and validation of a specific and reliable positive control for canine
ehrlichiosis molecular diagnostics. The gltA gene, a well-established target for E. canis
identification, was amplified using PCR with Taq polymerase, generating amplicons with
3' dA overhanging for TA cloning. After purification, the gltA gene was ligated into the
pTAPG3 vector using T4 DNA ligase, facilitating TA cloning. The ligation reaction
resulted in a visible, bright band, indicating successful incorporation of the gene into the
vector. Heat-shock transformation into competent E. coli DH5α cells, followed by plating
on Luria-Bertani with ampicillin agar dishes, yielded colonies for further screening.
Colony PCR was conducted, and a clear, distant band of the expected size (407 base pairs)
was observed in one transformant, affirming successful ligation and transformation.
Conversely, seven transformants displayed unexpected bands, indicating incomplete
ligation or the presence of undesired DNA fragments. The successful transformant was
subjected to plasmid isolation, and PCR analysis confirmed the presence of the gltA gene
insert within the plasmid. To ensure the authenticity of the obtained gene fragment,
sequencing was performed, resulting in 100% sequence identity with the gltA gene of
Ehrlichia canis, with no gaps observed. This successful sequencing further supported the
specificity and accuracy of the positive control. | en_US |
