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%.