Påvisande av Helicobacter spp hos hund : en metodologisk studie
The purpose of this study was to develop a reliable molecular-genetic method to determine the different species of helicobacter in dogs. The study is part of a larger project to map the prevalence of Helicobacter spp in healthy and sick dogs in Sweden, and to determine the possible connection of Hel...
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| Formato: | L3 |
| Lenguaje: | sueco Inglés |
| Publicado: |
SLU/Dept. of Animal Environment and Health (until 231231)
2007
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| Materias: |
| _version_ | 1855571811905830912 |
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| author | Nicol, Claire |
| author_browse | Nicol, Claire |
| author_facet | Nicol, Claire |
| author_sort | Nicol, Claire |
| collection | Epsilon Archive for Student Projects |
| description | The purpose of this study was to develop a reliable molecular-genetic method to determine the different species of helicobacter in dogs. The study is part of a larger project to map the prevalence of Helicobacter spp in healthy and sick dogs in Sweden, and to determine the possible connection of Helicobacter spp infection with gastrointestinal diseases in dogs.
Several published studies have reported on the prevalence of Helicobacter spp in dogs. The problem is that three of the most common species are so alike that a 16SrRNA-PCR with sequencing is not able to differentiate between them. In this study, DNA has been purified from samples and then a Multiplex PCR has been performed. Multiplex PCRs use multiple primers in one single PCR. The primers have been attached with fluorescent markers, and capillary electrophoresis is used to illustrate the results. In this way, very similar species can be differentiated from each other.
This paper also includes a short review of previous studies made in this field and the different methods used for diagnosis of infection with Helicobacter spp.
Samples from 17 dogs were collected and analyzed. Multiplex PCR was used on 12 healthy dogs. For the Multiplex PCR non-invasivly collected samples were used, namely faeces and mouth swabs. The remaining 5 dogs were patients with different gastrointestinal problems. From these dogs, in addition to faeces and mouth swab samples, biopsies were taken from the stomach and duodenum.
The study shows that the Multiplex PCR works well, but that the capillary electrophoresis is not able to illustrate the results. Also, all samples from all dogs were analyzed in a helicobacteria-specific 16SrRNA PCR and this showed Helicobacter spp in nearly all samples, the frequency of mixed helicobacter-infection being as common in mouth swabs as it was in faeces samples. This unexpected result further emphasises the need to develop the Multiplex PCR-technology, as normal sequencing cannot be used in severe mixed infections. Thus, the Multiplex PCR should prove to be a diagnostically reliable method, but further development and testing are needed. |
| format | L3 |
| id | RepoSLU11124 |
| institution | Swedish University of Agricultural Sciences |
| language | swe Inglés |
| publishDate | 2007 |
| publishDateSort | 2007 |
| publisher | SLU/Dept. of Animal Environment and Health (until 231231) |
| publisherStr | SLU/Dept. of Animal Environment and Health (until 231231) |
| record_format | eprints |
| spelling | RepoSLU111242017-09-28T08:18:38Z Påvisande av Helicobacter spp hos hund : en metodologisk studie Nicol, Claire Helicobacter spp canine helicobacter infektion metoder för diagnos av helicobacter spp infektion multiplex PCR kapillärelektrofores analys av icke-invasivt tagna prov för helicobacter spp infektion The purpose of this study was to develop a reliable molecular-genetic method to determine the different species of helicobacter in dogs. The study is part of a larger project to map the prevalence of Helicobacter spp in healthy and sick dogs in Sweden, and to determine the possible connection of Helicobacter spp infection with gastrointestinal diseases in dogs. Several published studies have reported on the prevalence of Helicobacter spp in dogs. The problem is that three of the most common species are so alike that a 16SrRNA-PCR with sequencing is not able to differentiate between them. In this study, DNA has been purified from samples and then a Multiplex PCR has been performed. Multiplex PCRs use multiple primers in one single PCR. The primers have been attached with fluorescent markers, and capillary electrophoresis is used to illustrate the results. In this way, very similar species can be differentiated from each other. This paper also includes a short review of previous studies made in this field and the different methods used for diagnosis of infection with Helicobacter spp. Samples from 17 dogs were collected and analyzed. Multiplex PCR was used on 12 healthy dogs. For the Multiplex PCR non-invasivly collected samples were used, namely faeces and mouth swabs. The remaining 5 dogs were patients with different gastrointestinal problems. From these dogs, in addition to faeces and mouth swab samples, biopsies were taken from the stomach and duodenum. The study shows that the Multiplex PCR works well, but that the capillary electrophoresis is not able to illustrate the results. Also, all samples from all dogs were analyzed in a helicobacteria-specific 16SrRNA PCR and this showed Helicobacter spp in nearly all samples, the frequency of mixed helicobacter-infection being as common in mouth swabs as it was in faeces samples. This unexpected result further emphasises the need to develop the Multiplex PCR-technology, as normal sequencing cannot be used in severe mixed infections. Thus, the Multiplex PCR should prove to be a diagnostically reliable method, but further development and testing are needed. Syftet med denna studie var att utveckla en molekylärgenetiskt pålitlig metod för att artbestämma helicobacterbakterier hos hund. Studien är en del av ett större projekt där man avser kartlägga helicobacterförekomsten hos friska och sjuka hundar i Sverige samt undersöka bakteriens betydelse för uppkomsten av gastrointestinala sjukdomar hos hund. Ett flertal studier har rapporterat om förekomst av Helicobacter spp hos hundar, men ett stort problem är att de allra vanligaste arterna är så pass lika varandra att en vanlig 16S rRNA-PCR (Polymerase Chain Reaktion) med sekvensering ej kan skilja dem åt. Detta arbete beskriver hur DNA har renats fram från provmaterial varpå en Multiplex-PCR metod med multipla primrar tillämpats i en enda PCR-reaktion. Dessa primrar innehåller fluorescerande ämnen som kan åskådliggöras med hjälp av kapillärelektrofores. Härigenom kan arter som liknar varann skiljas åt. Arbetet ger även en kort inblick i tidigare studier som gjorts inom området, samt de olika metoder som finns för diagnos av infektion med Helicobacter spp. Prov från 17 hundar analyserades och Multiplex-PCR utfördes på 12 friska hundar. Till Multiplex-PCR-reaktionen användes icke-invasivt tagna prover, faecesprov samt munskrapprov. De övriga fem hundarna var patienthundar som inkom till kliniken för olika gastrointestinala störningar. Från dessa hundar analyserades, förutom faeces- och munskrapprov, även biopsier från magsäck och duodenum. Resultaten visar att Multiplex-PCR-reaktionen fungerar bra men att kapillärelektroforesen ej kan åskådliggöra resultatet. Sammanfattningsvis kommer Multiplex-PCR vara en diagnostiskt pålitlig metod men vidare utveckling och studier krävs. Samtliga prover från samtliga hundar analyserades i en helicobacterspecifik 16S rRNA PCR och det visade sig finnas Helicobacter spp hos nästan alla de hundar som provtogs. Dessutom var frekvensen blandinfektion i munprov liknande den som ses i faecesprov, vilket ej var förväntat. Detta accentuerar ytterligare behovet av att utveckla Multiplex-PCR metoden då vanlig sekvensering inte kan användas vid kraftiga blandinfektioner. SLU/Dept. of Animal Environment and Health (until 231231) 2007 L3 swe eng https://stud.epsilon.slu.se/11124/ |
| spellingShingle | Helicobacter spp canine helicobacter infektion metoder för diagnos av helicobacter spp infektion multiplex PCR kapillärelektrofores analys av icke-invasivt tagna prov för helicobacter spp infektion Nicol, Claire Påvisande av Helicobacter spp hos hund : en metodologisk studie |
| title | Påvisande av Helicobacter spp hos hund : en metodologisk studie |
| title_full | Påvisande av Helicobacter spp hos hund : en metodologisk studie |
| title_fullStr | Påvisande av Helicobacter spp hos hund : en metodologisk studie |
| title_full_unstemmed | Påvisande av Helicobacter spp hos hund : en metodologisk studie |
| title_short | Påvisande av Helicobacter spp hos hund : en metodologisk studie |
| title_sort | påvisande av helicobacter spp hos hund : en metodologisk studie |
| topic | Helicobacter spp canine helicobacter infektion metoder för diagnos av helicobacter spp infektion multiplex PCR kapillärelektrofores analys av icke-invasivt tagna prov för helicobacter spp infektion |