Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction.
In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-infected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0.5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.
Drosten
Christian
C
Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Virology/Molecular Diagnostics, Bernhard-Nocht Strasse 74, 20359 Hamburg, Germany. drosten@bni-hamburg.de
Müller-Kunert
Ewald
E
Dietrich
Manfred
M
Gerdes
Johannes
J
Schmitz
Herbert
H
D000328:Adult; D004279:DNA, Viral / Q000032:analysis*; D020566:Dendritic Cells, Follicular / Q000166:cytology; D005629:Frozen Sections; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008198:Lymph Nodes / Q000166:cytology / Q000821:virology*; D008297:Male; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016662:Virus Integration / Q000235:genetics
D004279:DNA, Viral