Chapter 28
PROTOCOL
In Situ Detection of Viral Nucleic Acids
by Chemiluminescence
MONICA MUSIANI, PATRIZIA PASINI, ALDO RODA,
and MARIALUISA ZERBINI
Introduction
Chemiluminescence is the release oflight produced by a chemical reaction
from particular chemically excited molecules as a result of the transition
from an electronically excited state to a lower energy state, usually the
ground state. This phenomenon has provided the biologist with a unique
analytical tool in various analytical techniques in which small amounts of
analytes can be detected and quantified by measurement of the light
emitted by chemiluminescent reactions (Martin et al. 1995, Lorimier et
al. 1996, Erhardt et al. 1996, Girotti et al. 1996, Kricka LJ 1998, Ross et
al. 1999; Guerasimova et al. 1999; Roda et al. 1999). In the last few years,
it has been demonstrated that chemiluminescent detection systems which
combine the use of an enzyme and a substrate that is converted to a lightemitting product, have the highest potential sensitivity; moreover chemiluminescent systems can give precise and accurate quantitative results
since the luminescent signal intensity is proportional to enzyme concentration present in the reaction (Lamarcq et al. 1995).
Different chemiluminescent substrates for alkaline phosphatase (AP),
based on adamantyl 1,2-dioxetane phenyl phosphate derivatives, are now
commercially available [e.g. CSPD (PE-Tropix, Bedford, MA, USA) and
Lumi-Phos Plus (Lumigen, Southfield, MI, USA)]. These substrates allow
~ Monica Musiani, University of Bologna, Department of Clinical and Experimental
Medicine, Division of Microbiology, Via Massarenti 9, Bologna, 40138, Italy
(phone +39051-302435; fax +39051-341632; e-mail musiani@med.unibo.it)
Patrizia Pasini, University of Bologna, Department of Pharmaceutical Sciences,
Via Belmeloro 6, Bologna, 40126, Italy
Aldo Roda, University of Bologna, Department of Pharmaceutical Sciences,
Via Belmeloro 6, Bologna, 40126, Italy
Marialuisa Zerbini, University of Bologna, Department of Clinical and Experimental
Medicine, Division of Microbiology, Via Massarenti 9, Bologna, 40138, Italy
PROTOCOL
In Situ Detection of Viral Nucleic Acids
by Chemiluminescence
MONICA MUSIANI, PATRIZIA PASINI, ALDO RODA,
and MARIALUISA ZERBINI
Introduction
Chemiluminescence is the release oflight produced by a chemical reaction
from particular chemically excited molecules as a result of the transition
from an electronically excited state to a lower energy state, usually the
ground state. This phenomenon has provided the biologist with a unique
analytical tool in various analytical techniques in which small amounts of
analytes can be detected and quantified by measurement of the light
emitted by chemiluminescent reactions (Martin et al. 1995, Lorimier et
al. 1996, Erhardt et al. 1996, Girotti et al. 1996, Kricka LJ 1998, Ross et
al. 1999; Guerasimova et al. 1999; Roda et al. 1999). In the last few years,
it has been demonstrated that chemiluminescent detection systems which
combine the use of an enzyme and a substrate that is converted to a lightemitting product, have the highest potential sensitivity; moreover chemiluminescent systems can give precise and accurate quantitative results
since the luminescent signal intensity is proportional to enzyme concentration present in the reaction (Lamarcq et al. 1995).
Different chemiluminescent substrates for alkaline phosphatase (AP),
based on adamantyl 1,2-dioxetane phenyl phosphate derivatives, are now
commercially available [e.g. CSPD (PE-Tropix, Bedford, MA, USA) and
Lumi-Phos Plus (Lumigen, Southfield, MI, USA)]. These substrates allow
~ Monica Musiani, University of Bologna, Department of Clinical and Experimental
Medicine, Division of Microbiology, Via Massarenti 9, Bologna, 40138, Italy
(phone +39051-302435; fax +39051-341632; e-mail musiani@med.unibo.it)
Patrizia Pasini, University of Bologna, Department of Pharmaceutical Sciences,
Via Belmeloro 6, Bologna, 40126, Italy
Aldo Roda, University of Bologna, Department of Pharmaceutical Sciences,
Via Belmeloro 6, Bologna, 40126, Italy
Marialuisa Zerbini, University of Bologna, Department of Clinical and Experimental
Medicine, Division of Microbiology, Via Massarenti 9, Bologna, 40138, Italy