Detection of CCHFV in Dermacentor sp. andRhipicephalus sp. ticks using Real Time RT-PCR based on SYBR
Green
In the present study specific primer sets for each of the six known
CCHFV genotypes and one degenerate primer pair for the detection of all
genotypes, described by others were used
(Sas et al., 2018). These genotypes were
assigned to different geographic areas: I – West Africa, II – Central
Africa, III – South and West Africa, IV – Asia and Middle East, V-
South and East Europe, VI – Europe. The region targeted by these
primers was from nucleotide 1068 to 1248 for the S-sequence. Viral RNA
was transcribed to cDNA using SuperScript IV Reverse Transcriptase (ref.
18090050, Invitrogen) reagent kit and a 6-mer random primer (ref.
N8080127, Invitrogen).
One µl of DNAc, 0.2 µl of each CCHF-deg primer, 0,2 µl of each
genotype-specific CCHF-primer, 10 µl SybrGReen reagent and 8.6 µl H2O
were used, in a total reaction volume of 20 µl. The real time RT-qPCR
was performed with Roche LightCycler 96 System (Roche Diagnostics,
Germany). The cycling conditions used as follows: 95°c for 300 s
preincubation, followed by 45 cycles at 95°C for 10s (denaturation),
60°C for 10 s (annealing) 72°C for 30 s and 37°C for 30 s (cooling), the
annealing temperature being adjusted at 60°C depending on the primers
temperature.
For regulatory reasons regarding bioterrorism, it was not possible to
use a positive control. A negative control nuclease-free water was used
to rule out cross contamination of reagents and surfaces. Analysis of
the fluorescence data was conducted with the LightCycler 96 software.
Fifteen µl of PCR product was mixed with 3 µl of loading buffer and then
electrophoresed on 2% agarose gel in Tris-acetate-EDTA buffer.
Statistical analysis : The upper range of prevalence was
calculated using VS Outbreak SurveillanceToolbox. The maximal prevalence
of viral infection in ticks was estimated assuming a sensitivity of
100% and a confidence level of 95%.