Figure 5. Evaluation of PCLSR.

Panel (A) fluorescent curves observed after PCLSR in MX3005 P real-time PCR system (Stratagene). The presence of relative fluorescence increase (‘R’-T) during the following minutes of the reaction was considered as positive result. The curves were observed starting from 32 min. (21^st case) up to 78 min. (64^th case). Panel (B) melting curve analysis of PCLSR products positive for ASFV’ DNA. The common temperature point for all positive samples is 85.3 °C. Panel (C) PCLSR – assessment of ASFV positive samples after addition of 1 μl of a 1:10 stock dilution of 10,000 x DMSO concentrated SYBR Green I dye (Invitrogen) to the reaction tubes and UV light illumination. Panel (D) gel electrophoresis of (1.5% agarose gel, stained with a SimplySafe solution) (EURx, Gdansk, Poland) of PCLSR products. Visible “ladder-like” pattern in ASFV positive samples. Panel (E) PCR for PCLSR results verification. The PCR was conducted with application of outer-spiral primers. Field samples from 17 ASF cases in wild boar and 3 outbreaks in pigs (lanes 1–20), 21 – blood collected from a non-infected pig, 22 – cDNA extracted from a classical swine fever virus (CSFV) strain Alfort/187, 23 – positive control standard ASFV plasmid containing a p72 gene fragment (7.2 × 10⁷ copies μl⁻¹); 24 – cDNA extracted from a porcine reproductive and respiratory syndrome virus (PRRS) strain Lelystad (genotype I). M – molecular length marker GeneRule 100 bp DNA Ladder Plus (Thermo-scientific, Waltham, Massachusetts, USA).