Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli.

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/μl 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.

[1]  K. Livak,et al.  Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. , 1995, PCR methods and applications.

[2]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[3]  B. Ray,et al.  Single copy Babesia microti hsp70. , 1996, Molecular and biochemical parasitology.

[4]  S. Little,et al.  Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene. , 1992, American journal of human genetics.

[5]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[6]  E. Lavy,et al.  Coinfection with multiple tick-borne and intestinal parasites in a 6-week-old dog. , 2007, The Canadian veterinary journal = La revue veterinaire canadienne.

[7]  C. Colitz,et al.  Coinfection with Multiple Tick-Borne Pathogens in a Walker Hound Kennel in North Carolina , 1999, Journal of Clinical Microbiology.

[8]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[9]  K. Pfister,et al.  Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil , 2007, Parasitology Research.

[10]  Thomas Emrich,et al.  A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples , 2008, Medical Microbiology and Immunology.

[11]  H. Iseki,et al.  Development of TaqMan-based real-time PCR assays for diagnostic detection of Babesia bovis and Babesia bigemina. , 2007, The American journal of tropical medicine and hygiene.

[12]  J. Wengel,et al.  The solution structure of a locked nucleic acid (LNA) hybridized to DNA. , 1999, Journal of biomolecular structure & dynamics.

[13]  G. Kowalchuk,et al.  Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ , 2007, BMC Genomics.

[14]  S. Telford,et al.  A subtropical case of human babesiosis. , 1997, Acta tropica.

[15]  D. Swinkels,et al.  Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes) , 2002, Human mutation.

[16]  R. Ganta,et al.  Multiplex Detection of Ehrlichia and Anaplasma Pathogens in Vertebrate and Tick Hosts by Real‐Time RT‐PCR , 2006, Annals of the New York Academy of Sciences.

[17]  K. Pierce,et al.  Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[18]  L. Sariya,et al.  Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood. , 2009, The Southeast Asian journal of tropical medicine and public health.

[19]  O. Opare-Sem,et al.  Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions. , 2008, Journal of virological methods.

[20]  M. Bergeron,et al.  Multiplex Real-Time PCR Assay for Detection of Influenza and Human Respiratory Syncytial Viruses , 2004, Journal of Clinical Microbiology.

[21]  Yi-Wei Tang,et al.  Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. , 2005, The Journal of molecular diagnostics : JMD.

[22]  D. Raoult,et al.  Detection of ehrlichiae in African ticks by polymerase chain reaction. , 2000, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[23]  Ofer Peleg,et al.  Use of Chimeric DNA-RNA Primers in Quantitative PCR for Detection of Ehrlichia canis and Babesia canis , 2009, Applied and Environmental Microbiology.

[24]  Russell Higuchi,et al.  Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions , 1993, Bio/Technology.

[25]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.