Diagnosis of plant viruses by nucleic acid hybridization

Authors

• Reijo Karjalainen Department of Plant Pathology, University of Helsinki SF-00710 Helsinki, Finland
• Leo Rouhiainen Department of Microbiology, University of Helsinki SF-00710 Helsinki, Finland
• Hans Söderlund Orion Genetic Engineering Laboratory, Valimotie 7 SF-00380 Helsinki, Finland

DOI: https://doi.org/10.23986/afsci.72262

Abstract

Nucleic acid hybridization is a powerful technique for the diagnosis of many plant viruses not easily detected by serological techniques. It is particularly effective in the detection of viruses occurring in low amount in plant tissue, viruses that are poor immunogens or contain satellites. Molecular probes with desired specificities can be prepared by recombinant DNA techniques for large scale use. cDNA probes of potato virus X(PVX) RNA were made by molecular cloning, and the clones were 32P labelled by nick translation. Hybridization of cDNA to PVX RNA revealed 1 ng of purified virus in 2 µl spots dried onto nitrocellulose filter. Infected samples of crude leaf extracts were easily detected by hybridization, while probes did not react with healthy leaf samples. Nucleic acid hybridization research aims at replacing radiometric probes with nonradioactive methods involving enzymes which are directly or indirectly coupled to the probe and whose presence is observed with the aid of a colour changing substrate. Hybridization assay formats that can easily be automatized are under development. Sandwich hybridization is a simple test format developed for analyzing unpurified biological material, and it appears to be a powerful tool for microbial diagnostics. Sensitivity can be improved by using detection systems in which the specific activity of the probe is increased. Procedures such as ’polymerase chain reaction’, in which the amount of detectable nucleic acid sequences can be increased, are promising alternatives for increasing sensitivity. It is concluded that even if probe-based assays are in their infancy, they will no doubt develop towards such easy use as have immunological test kits.