Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa ; Department of Biology, National University of Lesotho, Roma, Lesotho.
PLoS One. 2013 Oct 17;8(10):e75827. doi: 10.1371/journal.pone.0075827. eCollection 2013.
A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.
基于cox III 基因的定量实时 PCR（qPCR） assay 用于检测和区分南非和莫桑比克水牛和牛血液样本中的 Theileria 种，使用熔解曲线分析。将获得的结果与用于混合感染的同时检测和区分 Theileria spp.的反向线印迹（RLB）杂交测定法进行比较，并与用于特异性检测 Theileria parva 的 18S rRNA qPCR 测定法结果进行比较。cox III 测定法检测到 Theileria parva、Theileria sp.（水牛）、Theileria taurotragi、Theileria buffeli 和 Theileria mutans。从分析的任何样本中均未检测到 Theileria velifera。17%的样本具有非特异性的熔解峰，4.5%的样本为阴性或低于测定法的检测限。cox III 测定法鉴定出比 RLB 测定法更多的 T. parva 和 Theileria sp.（水牛）阳性样本，并且还比 18S 测定法检测到更多的 T. parva 感染。但是，只有少数样本对良性 Theileria spp.呈阳性。据我们所知，T. taurotragi 从未从非洲水牛中鉴定出来，qPCR 测定法在一些样本中鉴定出它是出乎意料的。由于这些结果存在差异，因此对 cox III qPCR 产物进行了克隆和测序。序列分析表明，良性 Theileria spp.的 cox III 基因探针靶区存在广泛的种内和种间变异，因此解释了它们的低检测率。cox III 测定法特异性检测牛和水牛中的 T. parva 感染。本研究产生的序列数据可用于开发更具包容性的测定法，用于检测和区分水牛和牛的轻度致病和良性 Theileria spp.的所有变体。
Zotero 插件
