Comparison of real-time polymerase chain reaction with the conventional PCR assay for the diagnosis of Theileria parva in South Africa

[摘要] Theileria parva (T. parva) is transmitted from carrier buffalo to cattle causingCorridor disease in cattle. The 989/990 conventional Polymerase ChainReaction (PCR) assay used for the detection of T. parva is labour-intensive andhas the potential for contamination due to the need for post-amplificationhandling. Real-time PCR offers a way of addressing these limitations. Thisthesis describes the development of a TaqMan assay for the detection ofT. parva and a comparison between this real-time assay with the real-timeHybridization probe assay and the conventional PCR assay for the diagnosis ofT. parva.Theileria general forward and reverse primers and a T. parva TaqMan probespecific for the recognition of a conservative region of the T. parva 18S rRNAgene was designed. The TaqMan PCR assay could detect T. parva DNA at a2x10-5% parasitaemia with a 93% certainty. The primer pairs and probe onlycross-reacted with Theileria sp. (buffalo) and no amplification with otherTheileria species, bacteria or related haemoparasites was observed. Theileriasp. (buffalo) is genetically closely related to T. parva. However, its biology anddisease relations are not known. The TaqMan probe assay detected 87% of allpositive samples for evidence of the diagnostic sensitivity and 100% of allnegative samples tested negative for the diagnostic specificity assay.These results were compared with those obtained from 989/990 conventionalPCR and BioPAD Hybridization probe PCR which targeted the same gene.The Hybridization probe PCR appeared to be more sensitive than the TaqManprobe PCR or conventional PCR assay. With the specificity test, theHybridization probe PCR proved to be more specific than the other two assays.All three tests gave similar results for the diagnostic specificity. The TaqManprobe assay with its high sensitivity, wide range of detection ability andsimplicity is particularly useful in the detection of T. parva. However, furtherstudies are required to improve the specificity of the TaqMan PCR assay inorder to eliminate the detection of Theileria sp. (buffalo).

[发布机构] University of the Free State