Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli
Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli
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DOI：
10.1016/j.vetpar.2010.06.039
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发表时间：
2010-10-29
影响因子：
2.6
通讯作者：
Harrus, Shirnon
中科院分区：
文献类型：
--
作者：
Peleg, Ofer;Baneth, Gad;Harrus, Shirnon
犬埃立克体和犬巴贝斯沃格利是两种具有世界性重要意义的硬虱传播的犬病原体。这两种病原体都是由棕狗扁虱Rhipicephalus sanguineus传播的，它在全球的分布越来越多。采用双标记探针，建立了同时检测犬棘球绦虫和犬沃氏杆菌的多重实时定量聚合酶链式反应(QPCR)方法。目的基因为犬E.canis的16S rRNA和犬热休克蛋白70(HSP70)。以犬β肌动蛋白(ACTB)基因作为内对照基因。该分析不使用任何预扩增步骤，如套式反应。在同一试管中其他扩增基因的模板浓度较高的情况下，在犬DNA存在的情况下，测试多重qPCR方法中每一种反应的敏感性。该多重检测方法的检测阈值为1-10拷贝/亩L，犬分支杆菌HSP70和ACTB的扩增不受其他同时反应的影响，而犬分支杆菌16S rRNA基因的扩增受干扰较小。这项测试将对诊断实验室有用，并可能节省时间、劳动力和成本。(C)2010爱思唯尔B.V.保留所有权利。
Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/mu l in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs. (C) 2010 Elsevier B.V. All rights reserved.