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Journal of Veterinary Diagnostic Investigation Vol. 21 Issue 5, 649-654
Copyright © 2009 by the American Association of Veterinary Laboratory Diagnosticians
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Full Scientific Reports

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

Yooncheol Ha and Chanhee Chae1

Correspondence: 1Corresponding Author: Chanhee Chae, Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, San 56-1, Shillim-dong, Gwanak-gu, Seoul, 151-742, Republic of Korea. swine{at}snu.ac.kr

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.

Key Words: Fixation time • in situ hybridization • polymerase chain reaction • porcine circovirus • postweaning multisystemic wasting syndrome • probe size






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