INTRODUCTION OF A FAST AND SENSITIVE FLUORESCENT IN-SITU HYBRIDIZATION METHOD FOR SINGLE-COPY DETECTION OF HUMAN PAPILLOMAVIRUS (HPV) GENOME

At present, in situ hybridization (ISH) is the only method for detecti on of specific genes in morphologically intact cells or tissue. We hav e developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to f ive copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotid es (Method 1). The entire procedure can be carried out in 4.5 hr throu gh the elimination of some of the steps routinely used in other ISH pr otocols. We also compared the sensitivity of this new FISH method (Met hod 1) to four other FISH techniques: digoxigenin-labeled DNA probe (M ethod 2); fluorescein-15-d-ATP labeled oligonucleotides (Method 3); fl uorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labele d probe (Method 5), for their ability to detect HPV DNA in the HPV-pos itive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copie s), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other m ethods employed. Method 1 was the only method that could reliably dete ct an HPV-16 genome in all SiHa cells. Our data suggest that the Metho d 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (inc luding HIV), oncogenes, and drug-resistant genes, in a variety of morp hologically intact cells and tissues.