Analysis of Methods for Screening Plant Viruses

Aw, Dennis Wei Jin (1987) Analysis of Methods for Screening Plant Viruses. PhD thesis, University of Glasgow.

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Potato virus X, belonging to the group of viruses known as the potexviruses, is an economically important virus, affecting potato crops worldwide. The detection of this and other plant viruses has, so far, mainly been dependent upon the antigen-antibody assays such as the enzyme-linked immunosorbent assay (or ELISA). By using the ELISA: 1) a large number of samples can be handled at the same time; 2) the antigen-antibody reaction is specific for the virus; 3) results can be obtained within a couple of hours using prepared trays, 4) the assay uses an immunoglobulin marker in place of a radiolabelled isotope, resulting in negligable loss of activity after extended periods of time, and 5) the assay is sensitive with small amounts of enzyme-labelled immunoglobulin, thereby dispensing with expensive detection systems. The use of the ELISA for the detection of PVX strains in crude plant sap showed a high degree of sensitivity. The assay could detect isolates of both strain-group 3 (PVX/PVX N) and strain-group 2 (PVB) to at least 10 x dilution (reaching 10 x dilution with one isolate of strain-group 3). The assay was also very sensitive in the detection of purified PVX/PVB viral particles, detecting 20 ng of viral particles or 200 pg RNA with purified RNA samples (equivalent to approximately 33 ng virus particles). However, the main disadvantage of using serological assays, even with one as sensitive as the ELISA, is that only viral antigens are detected, giving no information about the infectious RNA within. An alternative system was, therefore, developed with the aim that it would be as sensitive as the ELISA, easy to handle, and one which allowed the detection of the infectious viral RNA. The assay was a dotblot system using nitrocellulose to which the RNA was bound by baking. Aliquots of infected sap, purified viral particles or RNA were applied and bound to sheets of nitrocellulose. The filter, containing the samples, was then be hybridised with labelled complementary DNA probes, detecting the RNA. Two systems were devised for the hybridisation stage of the assay using two different probes, one radiolabelled and the other a biotinylated probe. Both 32P-labelled and biotinylated nucleotides were incorporated into cDNA probes, using PVX N RNA as template. These probes hybridised to complementary sequences, detecting the signals on the filters by autoradiography for the 32P-labelled probe and with an enzyme reaction (streptavidin conjugated to biotinylated alkaline phosphatase) for the the biotinylated probe. The alkaline peroxidase-conjugated streptavidin, in the presence of substrates, converted the substrates into visible precipitates on the filters. With the radiolabelled probe, RNA in the viral particles of crude sap could only be detected to 10-1 x dilution with both PVB and PVX isolates (reaching 10-2 x dilution maximum), compared to 10-3 to 10-6 obtained with the same sample using ELISA. For the detection of purified viral particles, the radiolabelled probe was sensitive to 20 ng and to 200 pg with purified RNA. Results for the biotinylated probe were identical, although the radiolabelled probe could be increased in sensitivity when the exposure time to X-ray film was increased. A third probe, produced by cDNA cloning a section of the PVX N RNA template and labelled by nick translation, was also used to detect complementary RNA on filters. The final clone (pPVX21 N) used for hybridisations contained an insert between 550-600 bp in length but only detected PVX RNA in the PVX (strain-Group 3, PBI) crude sap samples to 10 and purified RNA to 20 ng with a 7 day exposure, results which were much less sensitive than either of the cDNA probes by at least 2 logs. However, an increase of non-specific hybridisation was also noted with the filter. The nick-translated probe did not detect PVB (strain-Group 2, PBI) or PVX N in the crude sap isolates or purified viral particles at all, unlike the cDNA probes. In addition to the development of these assays for the detection of viral RNA, studies were undertaken to obtain structural information on the 3' terminal region of the RNA. These studies included the synthesis of complementary DNA from an RNA template in the presence and absence of various primers. Initial results conflicted with the published literature of Sonenberg (1978) which reported the absence of poly (A) sequences at it 3' terminus, but confirmed the report of a poly (A)-rich region in PVX RNA by Morozov et al. (1983). These results were checked by affinity chromatography on oligo d(T) cellulose columns which selectively bound poly (A)-rich regions in RNAs. These experiments were further backed by selectively degrading the RNA with enzymes which could then be sized on polyacrylamide gels. The position of the poly (A) sequences was determined using RNase H, an enzyme which specifically attacked and degraded these poly (A) nucleotide sequences when bound to oligo d(T), leaving the RNA in two or more fragments which could then be analysed by polyacrylamide gel electrophoresis. Lastly, the size and position of these poly (A) sequences were checked by direct RNA sequencing, a method dependent upon a series of base-specific RNases. PVX RNA was labelled, purified on a 4% polyacrylamide gel to remove excess label, digested with the enzymes and analysed on a thin gel by thin-layer, polyacrylamide gel electrophoresis and autoradiography. The size of the poly (A) region in PVX N RNA was found to be between 8 and 16 nucleotides long and positioned at the 3' terminus rather than being an internal sequence.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Botany, Plant pathology
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76658
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 13:57
Last Modified: 19 Nov 2019 13:57

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