Utilization of a rate enhancement hybridization buffer system for rapid insitu hybridization for the detection of porcine circovirus in cell cultureand in tissues of pigs with postweaning multisystemic wasting syndrome

A rapid in situ hybridization (ISH) technique for the detection of porcine circovirus (PCV) nucleic acid in cell culture and formalin-fixed paraffin-e mbedded tissues was developed. A fluorescein-labeled RNA probe was transcri bed from a plasmid containing 530 bp of the ORF1 of a PCV isolated from a p ig with postweaning multisystemic wasting syndrome (PMWS). Hybridization us ing standard hybridization buffer was performed at 42 C for 16 hours and wa s compared to hybridization using rate enhancement hybridization (REH) buff er at 67 C for 2 hours. Hybridization was detected with an alkaline phospha tase-conjugated antifluorescein antibody. Ln both cultured cells and tissue s from pigs with PMWS, the signal intensity and number of labeled cells in sections hybridized with REH buffer were equal to those of sections hybridi zed with standard hybridization buffer. The total time required for ISH usi ng the REH buffer is 7-8 hours, thus making this protocol suitable for appl ication in routine PCV diagnosis.