Thesis submitted in fulfilment of the requirements for the degree
Master of Technology: Biomedical Technology
in the Faculty of Health and Wellness Sciences
at the Cape Peninsula University of Technology, 2012 / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi. Routine water quality analysis however, does not include monitoring for viral contaminants, as this process is hampered by the lack of simple, reliable, time- and cost-effective testing methods to concentrate and detect viral pathogens. The primary aim of this study was thus to establish and optimise routine monitoring techniques for the detection of rota-, adeno- and enteroviruses in the Berg- and Plankenburg Rivers, Western Cape. Initially, various concentration and extraction methods were compared for the optimum recovery of viruses from spiked water samples. One hundred milliliter water samples were spiked with one milliliter rotavirus and two milliliters adenovirus control virions (Coris Bioconcept, Gembloux, Belgium). Optimisation testing of enterovirus was however, not completed due to the unavailability of a positive control. Four viral concentration techniques, namely the Silicon dioxide (SiO2) method, positively charged, negatively charged and the mixed-ester filters, were compared. Various nucleic acid extraction methods were also employed to establish which method would provide optimum yields for both DNA and RNA nucleic acids. The extraction techniques included the TRIzol method (Invitrogen, California, USA) for RNA extraction, the Roche High Pure PCR Template Preparation kit (Roche, Mannheim, Germany) for DNA extraction, and the QIAamp Ultrasens Virus kit (Qiagen GmbH, Hilden, Germany) for simultaneous RNA and DNA extraction. The use of virus specific primers within the PCR technique was also optimised. In addition, gene specific primers and oligo(dT)15 primers were tested and compared to establish which primers would yield the best results since gene specific primers are said to be more sensitive than oligo(dT)15 primers (van Pelt-Verkuil et al., 2008) when synthesising cDNA (rotavirus). The SiO2 concentration method yielded variable results when it was used with the various nucleic acid extraction techniques in this study, since positive PCR results were obtained when used in combination
III
with some techniques, while negative results were obtained with others. Similarly, variable results were also obtained when negatively charged filters were used to concentrate virus particles, and when this method was used in conjunction with various virus nucleic acid extraction techniques to identify different viruses by RT-PCR and PCR. Results for the non-charged mixed-ester filter were comparable to the positively charged filters when used in conjunction with the various nucleic acid extraction techniques in this study. Both these techniques yielded the highest viral particle concentration from the spiked water samples. Pilot study results indicated the presence of rotavirus and adenovirus detected by RT-PCR and PCR respectively, when filtering through the positively charged filter. The positively charged filter/QIAamp UltraSens virus kit combination was found to be the optimum combination when analysing the spiked water results and was then employed for the concentration of virus particles in the river water samples collected from the Plankenburg- and Berg River systems throughout the study period. The expected PCR product of 346 bp for rotavirus was absent in all 72 river water samples analysed for both river systems. In contrast to the PCR results obtained for rotavirus, the expected product of 261 bp for adenovirus was detected in 22 (30.5%) samples collected throughout the study period. Fifteen of the 22 adenovirus positive samples were found in the Plankenburg River (distributed over all sites), while seven of the 22 adenovirus positive samples were found in the Berg River (all sites). A nested PCR was used to detect enterovirus in the river water samples collected from both river systems throughout the study period. In the first round of the enterovirus PCR 15 river water samples (at various sites for both river systems) yielded a faint 513 bp product. Further amplification by nested PCR then yielded 13 (18.1%) positive nested PCR products of 297 bp. The incidence of adenovirus and enterovirus in river waters reported in the current study and the Van Heerden et al. (2003) investigation motivates for similar studies to be conducted in drinking water, dam water used for recreational purposes as well as rainwater, which is gaining popularity as a sustainable water source.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:cput/oai:localhost:20.500.11838/1497 |
| Date | January 2012 |
| Creators | Saayman, Michael John |
| Publisher | Cape Peninsula University of Technology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | http://creativecommons.org/licenses/by-nc-sa/3.0/za/ |
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