primers 1 u03bcl of reverse primers 1 5 u03bcl of 25 mm mg 2 2 0 u03bcl of 2 5 mm dntps 2 5 u03bcl of 10 u00d7 mg 2 free buffer 0 2 u03bcl of taq dna polymerase 11 8 u03bcl distilled water and 5 u03bcl template dna the pcr reaction started with an initial step of pre heating the pcr mixture at 95 u00b0 c for 5 minutes and amplification of dna was carried out by 38 cycles with the following parameters denaturing at 94 u00b0 c for 30 seconds annealing at 55 u00b0 c for 30 seconds and elongating at 72 u00b0 c for 30 seconds the pcr reaction was completed with a final extension step of 5 minutes at 72 u00b0 c and then kept at 4 u00b0 c pcr products were next analyzed on a 1 agarose gel and visualized by ethidium bromide staining in addition another pair of pcr primers used to detect pcv2 viremia has the following sequences pcv2 f2 forward and pcv2 r2 reverse primers were used to amplify a 676 bp fragment from pcv2 at 25 days after challenge piglets were sacrificed and anatomized to observe pathological abnormalities in organs and collect lymph nodes and lungs tissue samples were fixed in 4 formaldehyde.