Please cite this article in press as: Peleg, O., et al., Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli. Vet. Parasitol. (2010), doi:10.1016/j.vetpar.2010.06.039 ARTICLE IN PRESS G Model VETPAR-5390; No. of Pages 8 Veterinary Parasitology xxx (2010) xxx–xxx Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli Ofer Peleg a , Gad Baneth b , Osnat Eyal b , Jacob Inbar a , Shimon Harrus b,∗ a Genaphora LTD, 4 Habarzel St., Tel Aviv 69710, Israel b Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel article info Article history: Received 24 March 2010 Received in revised form 27 May 2010 Accepted 30 June 2010 Keywords: Multiplex qPCR Ehrlichia canis Babesia canis vogeli abstract Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a world- wide 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/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. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Quantitative real-time polymerase chain reaction (qPCR) is a reliable technique for quantitative analysis of specific DNA and RNA sequences (Higuchi et al., 1993). It is commonly used for quantification of gene expression, RNA interference, and pathogen detection. Simultaneous detec- tion and quantification of several pathogens in the same tube decreases the assays’ time labor and cost. However, this is often at the cost of reduction in sensitivity and accu- racy (Ferrie et al., 1992). The reduction in sensitivity can be acceptable if the assay quantifies gene expression with ample mRNA quantities. However, in the case of pathogen detection, this compromise could be critical due to the ∗ Corresponding author. Tel.: +972 8 9489022; fax: +972 8 9489956. E-mail address: harrus@agri.huji.ac.il (S. Harrus). necessity of detecting the lowest possible pathogen num- ber and the associated clinical implications of false negative results. There are two main approaches for quantitative pathogen detection using real-time PCR: one with non- specific probes such as SYBR green and the other with specific probes such as TaqMan ® probes (Livak et al., 1995) or molecular beacons (Tyagi and Kramer, 1996). When detecting pathogens, there is a tendency to prefer specific probes due to their elevated specificity. Specific probes, such as dual-labeled probes or MGB-TaqMan ® probes are the preferred candidates for multiplex qPCR assays (de Kok et al., 2002; Doyle et al., 2005; Ferrie et al., 1992). Several other approaches were developed in order to con- struct high sensitivity multiplex assays, such as the ligation probe-based system (van Doorn et al., 2007) and the Sep- tiFast hybridization probes (Lehmann et al., 2007). Ehrlichia canis and Babesia canis vogeli are two tick- borne canine pathogens with a worldwide importance. 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.06.039