Optimal probe size and fixation time for the detection of Porcine circovirus-2 DNA by in situ hybridization in formalin-fixed, paraffin-embedded tissue.

Probe size and fixation time for detecting Porcine circovirus-2 (PCV-2) by in situ hybridization in formalin-fixed, paraffin-embedded lymph nodes from experimentally infected pigs were optimized. In situ hybridization using a 169-base pair (bp) probe detected significantly fewer PCV-2-positive cells than when using 8 other larger probes (P < 0.05). The difference in hybridization intensity between smaller probes (169 and 225 bp) and larger probes (416, 473, 571, 631, 693, and 753 bp) was statistically significant (P < 0.05). The PCV-2-positive cells were consistently detected in lymph nodes fixed up to 3 days; thereafter, the number of positive cells declined. The PCV-2-positive cells were detected in lymph nodes fixed for up to 730 days. The difference in hybridization intensity between samples fixed for a short term (1 or 3 days) and a longer term (4-730 days) was statistically significant (P < 0.05). The data demonstrates that the optimal probe size and fixation time for detecting PCV-2 in formalin-fixed, paraffin-embedded lymph nodes is 473 bp and 1-3 days, respectively.