Figure 3: PCLSR parameters evaluation.

(A) PCLSR sensitivity test. Gel electrophoresis of resulted products. Serial 10-fold dilutions of a standard ASFV plasmid with p72 gene fragment (from 7.2 × 10⁷ – lane #1 copies, to 0.07 copies, μl⁻¹ – lane #9). The limit of detection (LoD) reached 7.2 × 10² copies, μl⁻¹ (lane #6). (B) PCLSR reaction time test. Incubation of reaction mixtures in time from 30 min. up to 90 min. The first presence of specific “ladder-like” products is observed after 45 min. (C) Specificity test. 1 – blood collected from a non-infected pig, 2 – positive control standard ASFV plasmid containing a p72 gene fragment (7.2 × 10⁷ copies μl⁻¹); 3 – blood collected from a non-infected wild boar; 4 –cDNA extracted from a classical swine fever virus (CSFV) strain Alfort/187; 5 – cDNA extracted from a porcine reproductive and respiratory syndrome virus (PRRS) strain Lelystad (genotype I); 6 – cDNA of field porcine epidemic diarrhea virus (PEDV-NVRI2015). (D) Restriction analysis of PCLSR products using DraI enzyme (Thermo-scientific, Waltham, Massachusetts, USA). 1 – The digestion of PCLSR products resulted in occurrence of 3 different products sizing 115 bp, 165 bp and 185 bp, respectively. 2 – positive control standard ASFV plasmid containing a p72 gene fragment (7.2 × 10⁷ copies μl⁻¹). The structures 1)-3) present 3 obtained products. M – molecular length marker GeneRuler 100 bp DNA Ladder Plus (Thermo-scientific, Waltham, Massachusetts, USA).