DESIGN OF OLIGONUCLEOTIDE PRIMERS AND A PROBE FOR THE DEVELOPMENT OF A REAL-TIME PCR TEST SYSTEM FOR THE DETECTION OF *THEILERIA ANNULATA

Tropical theileriosis is an intracellular pathogen. Theileria annulata is responsible for economic losses to the cattle industry. The aim of this study was to design oligonucleotide primers and a fluorescent probe for detection of T. annulata DNA and to test their efficiency in real time PCR. The 18S rRNA gene sequences from GenBank were aligned using MEGA 11. A conserved region of the sequences analyzed was chosen as the target for amplification. The primer pair Tan F/Tan R and Tan Probe were designed by Vector NTI Advance and in silico sequence analysis was performed to evaluate them. The BLASTn analysis showed that the whole target fragment was identical to the T. annulata entries obtained from the search. The selected oligonucleotides were further tested by conventional and real-time PCR. A standard PCR yielded a product consistent with the expected amplicon length of 111 bp . Real-time PCR showed positive amplification signals with Ct values of 13.74 and 24.72. The experiments demonstrated that the selected target and fluorescence identification were enhanced using the primer-probe pair under the applied conditions. Tan F/Tan R and Tan Probe can therefore be the basis for the development of a real-time PCR assay for tropical theileriosis. Further studies on more types of field samples are needed to assess its utility in veterinary diagnostics and surveillance.