| Summary: | 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).
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