PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections.
In situ hybridization (ISH) provides a means for identifying viral genomes in the context of tissue pathology. We have developed a specific and sensitive ISH probe for the detection of cytomegalovirus (CMV) DNA in formalin-fixed, paraffin-embedded tissue sections. Digoxigenin-11-dUTP was incorporated into a 435-base pair fragment of the CMV Major Immediate Early (MIE) gene with use of the polymerase chain reaction (PCR). Hybridized probe was detected by reaction with antidigoxigenin antibody coupled to alkaline phosphatase and chromogenic substrates. This method has detected CMV infection in routine clinical specimens from a variety of tissue types, including colon, kidney, liver, and stomach. Infection in cells with and without characteristic inclusions is revealed with this probe. The background is so low that single infected cells are detected unambiguously. No cross-hybridization was observed with cells infected with other viruses of Herpesviridae. This approach may be useful for producing probes for the detection of other viral genomes in tissue sections.
Wood
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NB
Molecular Biology Diagnostics Laboratory, Rush Presbyterian St. Lukes Medical Center, Chicago, Illinois 60612.
Sheikholeslami
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M
Pool
M
M
Coon
J S
JS
D001483:Base Sequence; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000302:isolation & purification*; D004076:Digoxigenin; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*
D005404:Fixatives; D015345:Oligonucleotide Probes; D005557:Formaldehyde; D004076:Digoxigenin