DOI: 10.5176/2382-5685_VETSCI13.20
Authors: Ronel Pienaar, Abdalla A. Latif, Ben J. Mans and Oriel M. M. Thekisoe
Abstract:
The real-time hybridization PCR assay that targets the 18S rRNA V4 hyper-variable region of T. parva can be affected by mixed infections with T. sp. (buffalo)-like parasites. Therefore, molecular markers independent of the 18S rRNA gene were investigated for diagnostic potential. This included the p67, p104 and Tpr genes that are unique to T. parva. A conventional and SYBR® Green touch-down PCR method was developed for each gene and 240 buffalo from the Kruger National Park were screened. Results from the protein-based genes as well as the hybridization assays were compared to the Hybrid II assay that was used as gold standard. The SYBR Green assays compared well with the conventional, hybridization and Hybrid II assays for the majority of negative and T. parva positive samples. Some positive samples were missed with the protein genes, possibly due to sequence variation in the primer regions. The protein genes did, however, detect additional Hybrid II positive samples diagnosed as negative by the hybridization PCR. These samples were all T. sp. (buffalo) positive and indicate possible suppression of PCR signal due to template competition from mixed infections in the hybridization assay. Independent markers can thus be useful for accurate diagnosis of T. parva infection, where mixed infections occur in buffalo and the optimization of a SYBR Green real-time PCR setup allows for more efficient diagnostics compared with conventional PCR.
Keywords: Theileria parva, protein genes, mixed infections, PCR, real-time SYBR Green polymerase chain reaction
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