SENSITIVE IN-SITU HYBRIDIZATION WITH CATALYZED REPORTER DEPOSITION, STREPTAVIDIN-NANOGOLD, AND SILVER ACETATE AUTOMETALLOGRAPHY - DETECTIONOF SINGLE-COPY HUMAN PAPILLOMAVIRUS

The usefulness of standard in situ hybridization for viral nucleic aci d detection is occasionally limited by its sensitivity limit of 10 to 50 copies per cell. A modified version of the recently described signa l amplification method, catalyzed reporter deposition (CARD), and its applications to formalin-fixed cells and tissue sections is presented. Deposition of the reporter is facilitated by using horseradish peroxi dase catalyzing the deposition of biotinylated tyramide on the locatio n of the probe target. The biotin accumulation created is usually dete cted with streptavidin-labeled enzymes or fluorochromes. In the presen t investigation, this step was replaced by streptavidin-Nanogold and c ombined with silver acetate autometallography. This resulted in deep-b lack precipitation at positive in situ hybridized reaction sites. The sensitivity of this new approach was tested with a biotinylated, genom ic probe specific for human papillomavirus (HPV)-16/18. SiHa cells, a cervical carcinoma-derived cell line with one to two HPV16 copies per cell, and 10 histologically confirmed cervical carcinomas were used fo r the study. All samples were previously HPV16 positive with solution polymerase chain reaction, but only two of the cervical carcinomas wer e positive with standard in situ hybridization with barely visible sig nals. When employing CARD-Nanogold, SiHa cells and 9 of 10 biopsies pr oved positive with marked signals. It is concluded that this nonisotop ic method can detect single viral copies in situ in routinely fixed ma terial and may have the potential to replace in situ polymerase chain reaction in many applications.