A COMPARISON OF RT-PCR, IN-SITU HYBRIDIZATION AND IN-SITU RT-PCR FOR THE DETECTION OF RHINOVIRUS INFECTION IN PARAFFIN SECTIONS

We describe an in-situ RT-PCR method for the amplification of rhinovir us (RV) in fixed, paraffin-embedded HeLa cells employed as a model for human respiratory epithelium. HeLa cells were infected in-vitro with inocula of rhinovirus-16 ranging from 10(2) to 10(6) 50% tissue cultur e infective doses (TCID50), incubated for 18 h then fixed and processe d into paraffin blocks. Sections of the cell preparation were subjecte d to standard RT-PCR, in-situ hybridisation (ISH) or in-situ RT-PCR us ing specific oligonucleotide primers or probes directed against the 5' non-coding region of RV RNA. RT-PCR was found to be capable of detect ing RV16 RNA in one 8 mu m-thick section of cells infected with the lo west virus titre. ISH using digoxigenin labelled oligonucleotide probe s located RV16 signal in the majority of HeLa cells al the highest vir us titre, but in few or no cells with the lowest virus titre. In contr ast, in-situ RT-PCR detected RV16 in the majority of cells infected wi th this amount of RV16. There was a slight loss of morphology and fine localisation associated with the in-situ thermal cycling process. How ever, the sensitivity of in-situ RT-PCR is comparable to standard RT-P CR and greater than ISH for the detection of RV. In-situ RT-PCR has wi de applications for sensitive localization of low copy viral and RNA s equences within cells to investigate the role of viruses in a variety of clinical conditions. (C) 1997 Elsevier Science B.V.