Comparison of real-time polymerase chain reaction with the conventional PCR assay for the diagnosis of Theileria parva in South Africa
Papli, Natasha Ektha
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Theileria parva (T. parva) is transmitted from carrier buffalo to cattle causing Corridor disease in cattle. The 989/990 conventional Polymerase Chain Reaction (PCR) assay used for the detection of T. parva is labour-intensive and has the potential for contamination due to the need for post-amplification handling. Real-time PCR offers a way of addressing these limitations. This thesis describes the development of a TaqMan assay for the detection of T. parva and a comparison between this real-time assay with the real-time Hybridization probe assay and the conventional PCR assay for the diagnosis of T. parva. Theileria general forward and reverse primers and a T. parva TaqMan probe specific for the recognition of a conservative region of the T. parva 18S rRNA gene was designed. The TaqMan PCR assay could detect T. parva DNA at a 2x10-5% parasitaemia with a 93% certainty. The primer pairs and probe only cross-reacted with Theileria sp. (buffalo) and no amplification with other Theileria species, bacteria or related haemoparasites was observed. Theileria sp. (buffalo) is genetically closely related to T. parva. However, its biology and disease relations are not known. The TaqMan probe assay detected 87% of all positive samples for evidence of the diagnostic sensitivity and 100% of all negative samples tested negative for the diagnostic specificity assay. These results were compared with those obtained from 989/990 conventional PCR and BioPAD Hybridization probe PCR which targeted the same gene. The Hybridization probe PCR appeared to be more sensitive than the TaqMan probe PCR or conventional PCR assay. With the specificity test, the Hybridization probe PCR proved to be more specific than the other two assays. All three tests gave similar results for the diagnostic specificity. The TaqMan probe assay with its high sensitivity, wide range of detection ability and simplicity is particularly useful in the detection of T. parva. However, further studies are required to improve the specificity of the TaqMan PCR assay in order to eliminate the detection of Theileria sp. (buffalo).