In situ polymerase chain reaction detection of varicella zoster virus in infected cells in culture.
Polymerase chain reaction (PCR) technology provides a means of identifying genes and studying their expression with specificity and precision. Combined with in situ hybridization (ISH), the amplified gene can be localized within cells. Cell gene sequences (human alpha-tubulin) were identified by PCR-ISH first in uninfected cells in culture, then in human blood mononuclear cells, and finally in sections of human liver. Varicella zoster virus (VZV) DNA was detected for the first time in virus-infected cells in culture by PCR-ISH, and compared to ISH alone. Efficient PCR-ISH was achieved by fixation of cells or tissue sections with 2% paraformaldehyde, by amplification under conditions of complete exposure of the samples to the reagents, and by detection of amplified products using non-radioactive digoxigenin-labeled oligonucleotide probes internal to the amplified DNA segment. PCR-ISH increased the detection of VZV DNA in infected cells 5-fold compared to ISH alone. Compared to ISH alone, PCR-ISH enhances significantly the detection of virus DNA in infected cells.
Mahalingam
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R
Department of Neurology, University of Colorado Health Sciences Center, Denver 80262, USA.
Kido
S
S
Wellish
M
M
Cohrs
R
R
Gilden
D H
DH
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D007668:Kidney; D008099:Liver / Q000378:metabolism / Q000821:virology*; D008214:Lymphocytes / Q000166:cytology / Q000821:virology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D014404:Tubulin / Q000032:analysis / Q000096:biosynthesis
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D014404:Tubulin