A Bead-Based Multiplex Assay for the Detection of DNA Viruses Infecting Laboratory Rodents

Abstract

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.

Viromes and surveys of RNA viruses in camel-derived ticks revealing transmission patterns of novel tick-borne viral pathogens in Kenya

“…After performing sequencing analysis, the remaining tick individuals (241 H. truncatum ticks, 78 H. rufipes ticks, and 17 H. dromedarii ticks) were used to investigate the prevalence of IFTV or MATV, BLTV4, LMTV, and BanToV using the PCR primers and probes designed according to viral RdRp or polyprotein sequences (Table S3). A bead-based assay was performed to allow multiple detection of viruses in one reaction according to a previous study [ 36 ] and detect viral RNA in each tick (Supplementary Methods). qRT-PCR was performed to determine the viral RNA copies in individual ticks and camel serum samples using the same primers and probes as those utilized in bead-based multiplex assays (Supplementary Methods).…”

Validation of Multiplex PCR and Serology Detecting Helicobacter Species in Mice

“…The multiplex PCR was performed in a final reaction volume of 12.5 µL comprising 1× Multiplex PCR Kit buffer (Qiagen, Hilden, Germany), containing 3 mM MgCl 2 , dNTP mix, 0.5× Q-solution and HotStartTaq DNA polymerase, 0.2 to 0.4 µM of each primer ( Table 2 ), and 1 µL of purified DNA. The reaction conditions were run as described earlier but using 40 cycles of amplification in a Mastercycler (Eppendorf, Hamburg Germany) [ 29 ]. The detection of amplicons was performed via hybridization reaction, adding 10 µL of PCR product to the bead mixture containing 33 µL of tetramethylammonium chloride (TMAC) hybridization solution (0.15 M TMAC, 75 mM Tris–HCl, 6 mM ethylen diamin tetraacetate (EDTA), 1.5 g/L Sarkosyl, pH 8), 7 µL of 1× TE and a mixture of 2000 probe-coupled beads.…”

“…As positive control, plasmid-DNA was extracted from a dam + , dcm + E. coli strain containing the selected bacterial target sequences (Eurofins, Ebersberg, Germany) as described before [ 29 ]. The copy number/unit mass was calculated by assuming that 1 bp weighs about 660 Da.…”

“…For each probe, MFI values in reactions with no PCR product added to the hybridization mixture were considered as background values. Net MFI values were computed by subtraction of 1.2 times the maximal background value plus 5 MFI [ 29 ]. All samples were applied in duplicates.…”

A highly multiplexed broad pathogen detection assay for infectious disease diagnostics

“…Multiplexed assays such as the MAGPIX [ 10 , 11 ] or multiplexed real-time PCR [ 12 – 15 ] can increase the number of targets being tested. For example, Munro and colleagues described a multiplexed PCR assay with detection on the MAGPIX or Luminex instruments capable of detecting multiple influenza viruses with performance similar to real-time PCR [ 11 ].…”