Background: Hepatitis E (HEV) is a small, non-enveloped virus that belongs to the viral genus Hepevirus. HEV is a positive sense single-stranded RNA virus and there is insufficient information regarding its replication. This is mainly because the virus has low capacity to grow in normally used cell cultures. Many specific strand assay detection studies have been done in order to understand more about HEV replication. Unfortunately, these assays have the disadvantage of giving false positive results. Aim: The aim of this project was to improve the positive strand assay to increase specificity and eliminate false positivity which is due to high sensitivity of the polymerase chain reaction (PCR). False positivity occurs as remains of transfected material in the cell are amplified. Method: The samples used in this project were swine samples from Sweden and a human sample (plasmid clone of genotype 1) from India. Negative samples, extracted positive samples and transcribed RNA positive sense samples were used. The methods applied were cDNA synthesis, exonuclease I and RNase treatments, DNA purification kits followed by first and nested PCR. Result: The results of this study indicated great improvement of the detection assay especially for the transcribed RNA samples. Best results were obtained at a final concentration of 1.5mM MgCl2 in the mastermix. Conclusion: Changing the concentration of MgCl2 appeared to have a great effect on PCR specificity. Improving detection assays is very essential as they can be applied in the research field and in public health centers either for diagnosis or tracking disease outbreaks.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-218747 |
| Date | January 2014 |
| Creators | Elkhalifa, Dina |
| Publisher | Uppsala universitet, Institutionen för kvinnors och barns hälsa |
| Source Sets | DiVA Archive at Upsalla University |
| Language | Swedish |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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