Advances in the use of nucleic acid probes in diagnosis of viral diseases of man

Abstract

A variety of methods are now available for the preparation and labelling of viral nucleic acids for use as probes in diagnostic virology. Some of these are assessed including the use of synthetic oligonucleotides in place of molecularly cloned nucleic acids, and alternatives to labelling with radioactive isotopes such as biotin, enzymes and fluorochromes. Dot blot, sandwich, indirect sandwich and in situ hybridization are covered, and examples given of the current use of nucleic acid probes in detection of human viral infections. The potential and limitations of nucleic acid hybridization are discussed in the light of these new methods.

The use of in situ hybridization in studies of viral disease

“…The researcher aiming to become familiar with in situ hybridization techniques is faced with an increasing variety of different probe types, probe labelling strategies, hybridization protocols and detection systems (see Chapter 1, also reviews by Coghlan et aI., 1985;Moench, 1987;Maitland et aI., 1987;Grodyet aI., 1987;Norval and Bingham, 1987;Hofler, 1987;Myerson, 1988). The optimal strategy for each particular application will vary depending on the origin and preparation of the tissue being probed, and the nature and location of the target nucleic acid.…”

“…We have defined in situ hybridization as the use of labelled nucleic acid probes to detect complimentary sequences in tissue sections, cellular preparations, or chromosome spreads. Protocols involving the use of non-cellular preparations (such as filter and sandwich hybridizations) to detect viral genomes are not included, but have been described in previous reviews (Bornkamm et at., 1983;Maitland et at., 1987;Norval and Bingham, 1987). While we have focused on the use of in situ hybridization in the study of human viral diseases, it should be noted that the technique has been extensively employed in the study of viral infections in other species, both as animal models of human disease and in the context of veterinary medicine (e.g.…”

Molecular Biology of Aging Part I: An Introduction to Laboratory Techniques of Molecular Biology

“…To examine a specific mRNA sequence it is essential to have a cDNA probe, the nucleotide sequence of which is complementary to mRNA. By labeling the phosphates of the cDNA through a variety of processes, 31 the probe will recognize only the mRNA of interest following hybridization. Hybridization is a process that permits the mRNA and labeled cDNA to anneal to each other.…”

Applications of nucleic acid probes in veterinary infectious diseases

“…Gillespie also presents an overview of different hybridization formats which have either been developed or are being developed. Readers are also referred to reviews on nucleic acid probes by Minson and Darby ( 1982 ), Edberg ( 1985 ), Kulski ( 1985 ), Pereira ( 1986 ), Norval and Bingham (1987), Paul ( 1988 ), and Tenover ( 1988and Tenover ( , 1989.…”