University of Pretoria, Malaria Parasite Molecular Laboratory, Institute for Sustainable Malaria Control
Finland, Faculty of Veterinary Medicine, Departement Food and environmental hygiene
Stageonderwerp 2006 - 2007
MLST Campylobacter: Breeding Campylobacter, Isolation of DNA, PCR, Elektroforese, sequencing
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Samenvatting eindwerk 2014-2015: Role of the sample weight in norovirus detection in oysters by using molecular techniques
Shellfish contaminated with viruses may form a risk towards human health. For example, raw oysters can contain noroviruses which can cause outbreaks of acute gastroenteritis. Viral gastroenteritis is an inflammation of the stomach and intestines and causes symptoms such as diarrhea, vomiting or nausea, which mostly disappear after one to three days.
In this study, oysters were examined for the presence of noroviruses. The aim was to find whether the weight of the samples, or the sample size, has an influence on the capability to detect the viruses. Therefore, 10 batches of each six oysters were examined, thus a total of 60 oysters. Each batch was divided into one and five oysters in order to make a comparison. Of all the oysters the digestive gland was cut out and cleaned, since it is in this organ the noroviruses usually accumulate. In case of the five oysters, the glands were pooled together and analyzed as a whole.
After the viruses were extracted from the glands using a proteinase K solution, RNA extraction was performed using a NucliSENS miniMag system. This extraction technique is based on magnetic particles containing silica which will extract the nucleic acids from the samples. To be able to check the efficiency of the extraction, a Mengo virus control was added.
Real-time reverse transcription PCR (RT-qPCR) was performed in order to detect the possibly present viruses. Two sets of primers were used, one to detect norovirus genogroup I (GI) and another one to detect norovirus GII. To make quantification of the positive samples possible, dsDNA was also added to each PCR run.
14 of the 19 samples were positive for GI. 5 of these 14 originated from one-oyster samples, the other 9 came from the five-oyster samples. Three samples were positive for both GI and GII, namely one from a one-oyster sample and two from five-oyster samples. Detection of norovirus GI and GII was thus effective in both types of samples, although the extraction efficiency showed a better mean result for the five-oyster samples. The amount of detected genome copies per gram of sample on the other hand showed slightly higher amounts in the samples containing just one oyster gland.
Samenvatting eindwerk 2006-2007: Multilocus sequence typing of Campylobacter jejuni
Campylobacter jejuni is one of the most common causes of human enteritis in the industrialized countries. This Gram-negative bacterium is widespread in the environment. Poultry, cattle, pigs, domestic pets, and wild birds carry campylobacters in their intestinal tracts. These host animals may be a reservoir for the strains that infect humans.
Multilocus sequence typing (MLST) is a molecular characterization technique for the characterization of bacteria based on the sequencing of seven housekeeping genes. Each of the alleles is assigned an allele number on the basis of those already present in the database. These seven allele numbers is then assigned a sequence type (ST) The STs of the isolates are grouped into clonal complexes, based on similarity to a central allelic profile.
In this study, 23 isolates of Campylobacter jejuni from Finnish bovine feces and meat out 2003 were typed. 18 strains were successful characterised and subdivided in nine different sequence types. This nine sequence types belonged to five clonal complexes. Four strains had a new sequence type and there were two new alleles sequenced. The most sequence types were associated with cattle isolates. MLST is a good typed method but not even fast and easy for each Campylobacter strain. Some of the strains contain PCR inhibitors and Campylobacter jejuni in bovine might have more variability as the strains isolated from other sources.
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