Department of Animal Health, NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Berreaga 1, Derio, Bizkaia 48160, Spain.
Parasit Vectors. 2012 Aug 13;5:171. doi: 10.1186/1756-3305-5-171.
The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered.
We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca.
The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures.
蜱传边缘无浆体牛寄生虫环形泰勒虫在许多热带和温带地区流行，包括梅诺卡岛（巴利阿里群岛，西班牙）。实时 PCR 广泛用于梨形虫的检测，但通常不考虑定量。
我们开发了一种用于检测和定量环形泰勒虫的实时定量 PCR（qPCR）检测方法，该方法包括内部扩增对照（IAC），以监测潜在抑制剂的存在。计算了特异性、灵敏度、精密度、线性范围和 PCR 效率，并评估了将定量循环（Cq）值转换为数量（Q）的不同方法。该检测方法能够检测（100%概率）和定量（线性响应）100 个基因拷贝，临床灵敏度设定为每微升血液 10 个环形泰勒虫。然后，该检测方法在平行分析的 141 份牛血样本中通过 Luminex®悬浮阵列进行了验证，显示了这里开发的 qPCR 检测方法在田间条件下检测和定量寄生虫的实用性。一旦验证，它就被用于在梅诺卡岛的一个农场的 8 个携带动物中监测环形泰勒虫的寄生虫血症全年。
开发的 qPCR 检测方法提供了一种可靠且简单的方法来定量环形泰勒虫的感染负荷，这对于研究携带动物作为感染源的作用，或评估治疗和控制措施的效果可能至关重要。
