title: Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus.
abstract: New methods are described for combined intracellular reverse transcription (RT) and polymerase chain reaction (PCR) using single primer pairs, with direct incorporation of digoxigenin-11-dUTP into amplificants (direct in situ RT/PCR). Routinely used fixatives and minimal pre-treatments were employed. Target sequences of measles virus nucleocapsid (N) and phosphoprotein genes were detected within measles virus infected Vero cells, both in suspension and in formalin-fixed sections, that had been treated by in situ reverse transcription and 30 cycles of direct in situ PCR. Uninfected cells, omission of Taq polymerase, and irrelevant primers were used as controls. Distribution of measles virus within infected cells was determined by in situ hybridisation and immunocytochemistry for measles virus N gene and protein, respectively. Confirmation of amplification within sections was by gel electrophoresis, Southern blotting and sequencing of extracted amplicons. In the majority of cases, measles-infected cells exhibited intense cytoplasmic signal after direct in situ PCR; this was not seen in uninfected cells or infected cells reacted either with irrelevant primers or without Taq polymerase. Unfixed cells in suspension required nested reaction. Measles-specific in situ hybridisation and immunocytochemistry gave an identical signal distribution in sections. Nuclear artifact occurred in some sections and was unpredictable, although it was greatest either in areas of cellular damage, following DNase predigestion, or with vigorous protease pre-treatment. In situ RT-PCR is feasible for measles virus in acutely infected cells both in sections and in suspension. Further work is required to improve the procedure and to eliminate artefactual nuclear signal.
authors.affiliation: Department of Medicine, Royal Free Hospital School of Medicine, Hampstead, London, UK.
authors.forename: R
authors.initials: R
authors.lastname: Ray
authors.forename: P J
authors.initials: PJ
authors.lastname: Cooper
authors.forename: R
authors.initials: R
authors.lastname: Sim
authors.forename: N
authors.initials: N
authors.lastname: Chadwick
authors.forename: P
authors.initials: P
authors.lastname: Earle
authors.forename: A P
authors.initials: AP
authors.lastname: Dhillon
authors.forename: R E
authors.initials: RE
authors.lastname: Pounder
authors.forename: A J
authors.initials: AJ
authors.lastname: Wakefield
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002522:Chlorocebus aethiops; D004587:Electrophoresis, Agar Gel; D010450:Endopeptidases / Q000378:metabolism; D005240:Feasibility Studies; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D008459:Measles virus / Q000235:genetics / Q000302:isolation & purification*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase; D014709:Vero Cells
chemical_list: D005404:Fixatives; D005557:Formaldehyde; D012194:RNA-Directed DNA Polymerase; D010450:Endopeptidases