Development of real-time RT-PCR for the detection of human sapovirus in foods

Sammanfattning: Food-poisoning is a major health problem and an estimated half a million Swedes are food-poisoned annually, with acute gastroenteritis as a consequence. One of the major causes of contaminated foods is related to food- and waterborne viruses. To be able to trace back the source of contaminant, the method of detecting viruses must be specific and sensitive. No standardized method for detecting foods for sapovirus exists today. The aim of the work described in this bachelor thesis is to implement and opti-mize a real-time RT-PCR method for the detection of all genogroups of human sapovirus in foods. The optimization involves modifications of an already existing assay. Three synthetic DNA templates, representing the different genogroups of human sapovirus, were amplified by real-time one-step RT-PCR. The modification of the RT-PCR assay involved testing TINA modified primers over a temperature gradient and primers used by Karolinska University Hospital. All genogroups of human sapovirus were detected, but there was no notable improvement using the TINA modified primers or the primers used by Karolinska University Hospital. The opti-mum temperature for the RT-PCR was determine to 60°C and was further used in the experiments. A monoplex assay was set up to test the specificity of the multiplex RT-PCR.The monoplex assay revealed that only the primers and probe for DNA template repre-senting genogroup V was specific against its target. Fecal samples from sapovirus positive patients, a gift from Karolinska University Hospital, were extracted and screened for genogroup V. All fecal samples were positive for sapovirus, whereof one sample was positive for genogroup V. Three synthetic single-stranded DNA (ssDNA) templates were used as positive controls, representing the target sequences for the different genogroups of human sapoviruses. Problems with dilution series of these led to an investigation where different diluents were tested, as well as heating the samples. The troubleshooting of the DNA templates was not solved. The real-time RT-PCR assay developed in this work did not have the same sensi-tivity as for Oka et al. (2006). The sensitivity of the assay must improve to be used for foods, and hopefully will this project serve as a pilot study for further investiga-tion and development of a RT-PCR assay for detection of human sapoviruses in foods.