RAPID IN-SITU HYBRIDIZATION TECHNIQUE FOR THE DETECTION OF RIBONUCLEIC-ACIDS IN TISSUES USING RADIOLABELED AND FLOURESCEIN-LABELED RIBOPROBES

In situ hybridization (ISH) is a useful diagnostic and research tool, but is also time consuming. This study was conducted to determine if a rate enhancement hybridization (REH) buffer, developed for membrane h ybridization, could be used to decrease hybridization time for ISH. Ti ssue from swine with an enteric disease produced by a swine coronaviru s, transmissible gastroenteritis virus (TGEV), was used as a model to standardize hybridization conditions for a rapid ISH technique. Small intestinal sections from pigs experimentally and naturally infected wi th TGEV were hybridized for various times at 52 degrees C and 70 degre es C with a radiolabelled or a fluorescein-labelled RNA probe in a sta ndard hybridization or a REH buffer. Viral RNA was detected in intesti nes from as early as 30 min of hybridization by using both buffers wit h the radiolabelled probe; however, the signal was stronger with the R EH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridizati on by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at ea ch hybridization time and hybridization temperature using both radiola belled and fluorescein-labelled probes. With the REH buffer, hybridiza tion signal intensity was greater at 70 degrees C than at 52 degrees C for both probes. The best results were obtained when small intestinal sections were hybridized at 70 degrees C for 2 h using a radiolabelle d or a fluorescein-labelled probe diluted in the REH buffer. The fluor escein-labelled RNA probe with REH buffer resulted in a minimal non-sp ecific signal when compared with the radiolabelled probe. These studie s demonstrated that the REH buffer can be used to decrease the time of ISH for the detection of viral RNA. This rapid ISH technique should h ave broad applications in the utilization oi probe technology in diagn ostics and research for the detection of target ribonucleic acids in s itu. (C) 1997 Academic Press Limited.