title: In situ polymerase chain reaction amplification of HIV-1 DNA in brain tissue.
abstract: A direct in situ polymerase chain reaction (IS-PCR) assay is described for the detection of HIV-1 proviral DNA in formalin fixed paraffin embedded brain tissue. Biotin-16-dUTP is incorporated during the PCR process and microwave pretreatment of tissue sections ensures that no non-specific incorporation into damaged or nicked genomic DNA occurs. Two methods are compared to detect the biotinylated amplified product, the use of an avidin-biotin-alkaline phosphatase complex (ABC) and the application of tyramide signal amplification (TSA) which allows both chromogenic and fluorescence detection. TSA detection enhances the sensitivity of IS-PCR, permitting fewer PCR cycles and preserving tissue morphology.
authors.affiliation: Department of Pathology, University of Edinburgh, Western General Hospital, UK.
authors.forename: P M
authors.initials: PM
authors.lastname: Strappe
authors.forename: T H
authors.initials: TH
authors.lastname: Wang
authors.forename: C A
authors.initials: CA
authors.lastname: McKenzie
authors.forename: S
authors.initials: S
authors.lastname: Lowrie
authors.forename: P
authors.initials: P
authors.lastname: Simmonds
authors.forename: J E
authors.initials: JE
authors.lastname: Bell
mesh_terms: D001921:Brain / Q000821:virology*; D004279:DNA, Viral / Q000302:isolation & purification*; D005557:Formaldehyde; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D008872:Microwaves; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
chemical_list: D004279:DNA, Viral; D005557:Formaldehyde