28 In Situ Detection of Viral Nucleic Acids by Chemiluminescence
357
colorimetric detection, thus permitting an easier evaluation of the sample
(Musiani et al. 1996b).
Chemiluminescence in situ hybridization allows the quantification of the
results; in fact results on smears ofCaski and HeLa cells (which are known
to contain 500-600 copies of HPV 16 DNA and 10-50 copies of HPV 18
DNA respectively) demonstrated that the luminescent signal changed
in proportion to the number of known viral genome copies per cell (Musiani et al. 1997). In addition, studying the presence of cytomegalovirus
DNA in infected cells, increasing values of emitted photons/cell corresponding to the presence of hybridized cytomegalovirus DNA, could
be found in cells fixed at 48, 60, 72 and 96 h after infection, following
the cytomegalovirus replication cycle (Musiani et al. 1996a).
With chemiluminescence in situ hybridization, since the positive signal is
considered the one above threshold values, an objective evaluation of the
results could be achieved without any training on the microscope to read
the slides and so, doubts about positive or negative results are minimized.
Chemiluminescence in situ hybridization offers a permanent record of the
reactions as all the images of the samples are stored in the computer and
these images can be printed or sent for an evaluation in other laboratories
using floppy disks or other computer networks.
In conclusion, the chemiluminescent in situ hybridization assay can be
a useful tool for a sensitive detection and study of specific genetic sequences inside the cells. The use of the chemiluminescent in situ hybridization assay, which can be applied to detect one or two target nucleic
acids in cell smears, archival frozen and paraffin embedded tissue samples, may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging
gene expression in cells, providing a strong standardization of the methods, reagents and samples.
Acknowledgements. The research cited in this review has been partly supported by CNR
Target Project on "Biotechnology". MURST (Ministero della Universita e della Ricerca
Scientifica e Tecnologica) and University of Bologna Funds for Selected Research Topics.
Quantification
of the data
Objectivity
of the evaluation
Digital imaging
of the results
357
colorimetric detection, thus permitting an easier evaluation of the sample
(Musiani et al. 1996b).
Chemiluminescence in situ hybridization allows the quantification of the
results; in fact results on smears ofCaski and HeLa cells (which are known
to contain 500-600 copies of HPV 16 DNA and 10-50 copies of HPV 18
DNA respectively) demonstrated that the luminescent signal changed
in proportion to the number of known viral genome copies per cell (Musiani et al. 1997). In addition, studying the presence of cytomegalovirus
DNA in infected cells, increasing values of emitted photons/cell corresponding to the presence of hybridized cytomegalovirus DNA, could
be found in cells fixed at 48, 60, 72 and 96 h after infection, following
the cytomegalovirus replication cycle (Musiani et al. 1996a).
With chemiluminescence in situ hybridization, since the positive signal is
considered the one above threshold values, an objective evaluation of the
results could be achieved without any training on the microscope to read
the slides and so, doubts about positive or negative results are minimized.
Chemiluminescence in situ hybridization offers a permanent record of the
reactions as all the images of the samples are stored in the computer and
these images can be printed or sent for an evaluation in other laboratories
using floppy disks or other computer networks.
In conclusion, the chemiluminescent in situ hybridization assay can be
a useful tool for a sensitive detection and study of specific genetic sequences inside the cells. The use of the chemiluminescent in situ hybridization assay, which can be applied to detect one or two target nucleic
acids in cell smears, archival frozen and paraffin embedded tissue samples, may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging
gene expression in cells, providing a strong standardization of the methods, reagents and samples.
Acknowledgements. The research cited in this review has been partly supported by CNR
Target Project on "Biotechnology". MURST (Ministero della Universita e della Ricerca
Scientifica e Tecnologica) and University of Bologna Funds for Selected Research Topics.
Quantification
of the data
Objectivity
of the evaluation
Digital imaging
of the results