A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.
The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.
Research Program Infection and Cancer, German Cancer Research Center, Heidelberg, Baden-Württemberg, Germany.
Daniela
D
Höfler
Microbiological Diagnostics, German Cancer Research Center, Heidelberg, Baden-Württemberg, Germany.
Werner
W
Nicklas
Microbiological Diagnostics, German Cancer Research Center, Heidelberg, Baden-Württemberg, Germany.
Petra
P
Mauter
Research Program Infection and Cancer, German Cancer Research Center, Heidelberg, Baden-Württemberg, Germany.
Michael
M
Pawlita
Research Program Infection and Cancer, German Cancer Research Center, Heidelberg, Baden-Württemberg, Germany.
Markus
M
Schmitt
D000818:Animals; D001483:Base Sequence; D004266:DNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004267:DNA Viruses / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D051379:Mice; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015345:Oligonucleotide Probes / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D012376:Rodent Diseases / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary
D015345:Oligonucleotide Probes