MST-School of Pure and Applied Sciences

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This collections contains bibliographic information and abstracts of Master theses and dissertation in the School of Pure and Applied Sciences held in Kenyatta University Library

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Browsing MST-School of Pure and Applied Sciences by Author "Abel, Yoas Sefasi"

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    Development and comparison of capture enzyme-linked immunosorbent assay and indirect immunofkuorescent test in detection of Nairobi sheep disease virus
    (2011-08-16) Waiganjo, Naomi Njoki; Abel, Yoas Sefasi; Machuka, Jesse; Kwapata, Moses
    Nairobi Sheep Disease (NSD) is caused by Nairobi Sheep Disease Virus (NSDV) of the genus Nairovirus of the Bunyaviridae family. The diagnosis of Nairobi Sheep Disease relies on the inoculation of tissue culture with suspensions of infected samples followed by identification of the virus using indirect immuno-fluorescent assay. The tests have a number of drawbacks including low specificity, visual reading of results which requires highly skilled expertise and the need for tissue culture facilities. This study was designed to develop capture ELISA in order to improve the diagnosis of Nairobi Sheep Disease in infected sheep. Nairobi Sheep Disease Virus was inoculated into baby hamster kidney (BHK-21) cells, harvested and purified through sucrose gradient method in an Ultra centrifuge at 4°C. The purified Nairobi Sheep Disease Virus was titrated to determine the best working titer for immunization of animals. The purified virus was subjected to IIFA test and fluorescence indicated the presence of Nairobi Sheep Disease Virus. The animals immunized were rabbits and goats which were used for production of antibodies for CELISA test. C-ELISA was set-up using anti-goat sera as the primary antibody, purified Nairobi Sheep Disease Virus as antigen and anti-rabbit sera as the secondary antibody. A 1:400 dilution was established as best dilution for true positive and negative samples. Twenty samples cryopreserved at -70°C, obtained from KARI were tested using both IIFA and C-ELISA test. The diagnostic specificity of the developed C-ELISA was estimated from 20 samples. Out of the twenty samples tested, four (20%) for Nairobi Sheep Disease Virus using C-ELISA and five (25%) were positive using IIFA. The four positive samples from the two tests were from the same samples. False positive (5%) samples were picked by IIFA, which was confirmed by tissue culture technique. The level of agreement between developed C-ELISA and IIFA used as a gold test was 95%, and the Kappa index was 0.86. The perfect agreement indicated by Kappa values is an indication that both tests can be used. However, C-ELISA is a better test in that it is more flexible and less subjective. The sensitivity and specificity of C-ELISA was estimated at 80% and 100% respectively. In conclusion, the high diagnostic specificity of the developed C- ELISA can be adapted to test a large number of samples over short periods of time. The test can be useful during outbreaks of Nairobi Sheep Disease without need for tissue culture facilities. The newly developed C-ELISA would facilitate epidemiological studies on Nairobi Sheep Disease infections and enable the diagnosis in the field.
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    Genetic transformation of sweet potato[ipomoea batatas(L.) lam] with nicotiana protein kinase 1 gene to enhance drought stress tolerance
    (2011-11-29) Abel, Yoas Sefasi; Machuka, Jesse; Kwapata, Moses
    Sweet potato [Ipomoea batatas (L.) Lam] is a valuable source of food and its tubers provide high levels of starch, lysine, vitamin A and ascorbic acid. Globally, sweet potato is one of the highest yielding crops with a yield potential of 30 tons/ha. However in Africa sweet potato yields are very low. The area under sweet potato in Africa is approximately 1,714,000 hectares with an annual production of about 5.5 million tons. Drought stress is one of the most significant abiotic constraints to sweet potato production in sub Saharan Africa. The application of biotechnology tools like genetic engineering for the improvement of important crop plants has been shown to have great potential. The objective of the study described herein was to genetically transform selected sweet potato cultivars with a tobacco Nicotiana Protein Kinase 1 gene which has been shown to confer drought stress tolerance in transgennc maize, yeast and Arabidopsis thaliana. Four orange-fleshed sweet potato cultivars namely KSP36, Ukerewe, Mayai and Carrot were used in this study. It was possible to regenerate plants from KSP36 and Ukerewe cultivars on three concentrations (0.5, 1.0 and 1.5 mg/L) of Indole-3 acetic acid (IAA) with average regeneration efficiencies of 8.3% and 6.2%, respectively. The concentration of 1.0 mg/L IAA was found to be the best (P<0.002) for regeneration of Ukerewe and KSP36 sweet potato cultivars. The study also established that the use of leaf or stem as explant had no significant (P<0.219) effect on regeneration efficiency of the tested cultivars. However it was confirmed (P<0.001) that regeneration efficiency is genotype-dependent in sweet potato since plant regeneration was only achieved in two of the four tested cultivars. The Agrobacterium tumefaciens strain EHA101 harbouring the binary vector plasmid pSHX004 with the NPKI transgene and bar selectable marker gene was used for genetic transformation of Ukerewe and KSP36 sweet potato cultivars. Putatively transformed callus and plantlets were screened by including 0.5 mg/L bialaphos in sweet potato culture media. Putative transformation efficiencies of 1.04% and 1.59% were achieved for KSP36 and Ukerewe, respectively. The putatively transformed sweet potato produced in.this study need to undergo drought stress tolerance assays and RT-PCR to assess expression of NPKI gene and actual drought stress tolerance. The regeneration efficiency was a very important factor influencing transformation efficiency. The findings of this study could provide new opportunities for producing drought stress tolerant sweet potato for sub Saharan Africa using genetic engineering.