PhD Thesis Demeter Zoltan

4.4 Electron microscopic investigation
Urine and feces samples were suspended 1:3 in distilled water, cleared by low speed
centrifugation, followed by 20 min at 9000 x g. The samples were prepared according to
the single-droplet negative staining technique (Harris, 2007) and examined at a JEM
1011 transmission electron microscope (JEOL, Japan).
4.5.1 Purification of the nucleic acid
4.5 Genetic investigations
Tissue samples were homogenized in 10 ml phosphate buffered saline (PBS) and
centrifuged at 1500 x g for 10 min. The nasal swabs were immersed in PBS solution
with 10 ml/L antibiotic and antimycotic component (Sigma, USA) and then centrifuged,
while the liquid samples (i.e. urine, serum) were only centrifuged. The viral nucleic acid
was isolated from the supernatants using the QIAamp viral RNA Mini Kit (Qiagen,
Germany) and High Pure Viral Nucleic Acid Kit (Roche, Switzerland), according to the
manufacturers’ instructions.
4.5.2 Primers
4.5.2.1 CDV
Primers were designed for diagnostic and for phylogenetic purposes as well. For
diagnostic purposes, primer pair “CDV-A” specific to a 409 bases long segment of the
conservative region of the L gene of the CDV genome was designed. Samples that
tested positive with these primers underwent another RT-PCR assay using the “CDV-B”
set of primers that partially encompassed the H gene of the CDV. The amplicons
produced by primer pair “CDV-B” were subsequently used for an RFLP-based strain
discriminating technique. The phylogenetic analysis of the Hungarian and other CDV
strains was based on the full nucleotide sequence of the H and F genes, determined by
using other sets of primers (Table 4). The sensitivity of the diagnostic PCRs using
primer pair “A” was determined by using serial dilutions of a vaccine virus strain with a
previously determined number of CDV RNA copies (10 5 ) (data not shown). Primers F
to K were used only for vaccine 3 (Table 2).
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