title: A comparison of RT-PCR, in-situ hybridisation and in-situ RT-PCR for the detection of rhinovirus infection in paraffin sections.
abstract: We describe an in-situ RT-PCR method for the amplification of rhinovirus (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 culture infective doses (TCID50), incubated for 18 h then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to standard RT-PCR, in-situ hybridisation (ISH) or in-situ RT-PCR using specific oligonucleotide primers or probes directed against the 5' non-coding region of RV RNA. RT-PCR was found to be capable of detecting RV16 RNA in one 8 microns-thick section of cells infected with the lowest virus titre. ISH using digoxigenin labelled oligonucleotide probes located RV16 signal in the majority of HeLa cells at the highest virus titre, but in few or no cells with the lowest virus titre. In contrast, in-situ RT-PCR detected RV16 in the majority of cells infected with this amount of RV16. There was a slight loss of morphology and fine localisation associated with the in-situ thermal cycling process. However, the sensitivity of in-situ RT-PCR is comparable to standard RT-PCR and greater than ISH for the detection of RV. In-situ RT-PCR has wide applications for sensitive localization of low copy viral and RNA sequences within cells to investigate the role of viruses in a variety of clinical conditions.
authors.affiliation: University Medicine, Southampton General Hospital, UK.
authors.forename: P J
authors.initials: PJ
authors.lastname: Bates
authors.forename: G
authors.initials: G
authors.lastname: Sanderson
authors.forename: S T
authors.initials: ST
authors.lastname: Holgate
authors.forename: S L
authors.initials: SL
authors.lastname: Johnston
mesh_terms: D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D016612:Paraffin Embedding; D010850:Picornaviridae Infections / Q000175:diagnosis / Q000473:pathology / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D012229:Rhinovirus / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase