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Bakalářská Práce MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV BIOCHEMIE Bakalářská práce Brno 2018 Monika Kopečková MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV BIOCHEMIE STANOVENÍ GENOSPECIES RODU BORRELIA ZÍSKANÝCH Z RŮZNÝCH OBLASTÍ ČESKÉ REPUBLIKY POMOCÍ DNA SEKVENACE Bakalářská práce Monika Kopečková Vedoucí práce: prof. RNDr. Omar Šerý, Ph.D. Brno 2018 Bibliografický záznam Autor: Monika Kopečková Přírodovědecká fakulta, Masarykova univerzita Ústav biochemie Název práce: Stanovení genospecies rodu Borrelia získaných z různých oblastí České republiky pomocí DNA sekvenace Studijní program: Biochemie Studijní obor: Biochemie Vedoucí práce: Prof. RNDr. Omar Šerý, Ph. D. Akademický rok: 2017/2018 Počet stran: 59 Klíčová slova: Borrelia burgdorferi; Ixodes ricinus; Lymská borelióza; sekvenování DNA Bibliographic Entry Author: Monika Kopečková Faculty of Science, Masaryk University Department of Biochemistry Title of Thesis: Determination of genespecies of the genus Borrelia obtained from various regions of the Czech Republic by DNA sequencing Degree Biochemistry programme: Field of Study: Biochemistry Supervisor: Prof. RNDr. Omar Šerý, Ph. D. Academic Year: 2017/2018 Number of Pages: 59 Keywords: Borrelia burgdorferi; Ixodes ricinus; Lyme disease, DNA sequencing Abstrakt Tato bakalářská práce pojednává o problematice Lymeské boreliózy. Jedná se o velmi závažné onemocnění způsobované bakterií rodu Borrelia. Hostitelem těchto bakterií jsou především klíšťata rodu Ixodes. V první části práce lze nalézt fakta jak o nemoci, tak o původcích i přenašečích. Cílem práce bylo zjistit pozitivitu klíšťat pomocí qPCR (Real-Time PCR) a následně pozitivní vzorky sekvenovat a zjistit tak zastoupení jednotlivých druhů. Celkem bylo otestováno 733 vzorků klíšťat získaných ze všech krajů České republiky. Bylo zjištěno, že 165 vzorků (22,5 %) tvoří vzorky pozitivní na Bbsl, u 114 vzorků byl zjištěn i konkrétní druh. Abstract This Bachelor thesis is about Lyme disease, which is serious illness caused by complex of bacteria Borrelia burgdorferi. Host of these bacteria is a tick of genus Ixodes. The first part of this thesis talks about the illness, bacteria and hosts. The aim was to test the positivity of the ticks within qPCR (Real-Time PCR). Then we were sequencing the positive samples and found out the species of Borrelia. There were 733 samples together and 165 (22,5 %) of them were positive. In 114 samples we were able to determine the genospecies of Borrelia. Poděkování Zde bych chtěla poděkovat v první řadě mé rodině, která je mi velkou oporou nejen při psaní této bakalářské práce, ale i během celé mé cesty životem. Dále bych chtěla poděkovat svému příteli, který mě také velmi podporuje a bez kterého bych nejspíš měla velký problém s ovládáním některých programů, které byly nezbytné k vypracování této práce. V neposlední řadě chci poděkovat prof. RNDr. Omaru Šerému, Ph.D. a firmě ELISABETH PHARMACON za vůbec umožnění pracovat na této velmi zajímavé a mně blízké problematice. Prohlášení Prohlašuji, že jsem svoji bakalářskou práci vypracoval(-a) samostatně s využitím informačních zdrojů, které jsou v práci citovány. Brno 27. dubna 2018 ……………………………… Monika Kopečková Obsah Obsah ................................................................................................................................................................ 9 Seznam použitých zkratek ............................................................................................................................... 11 Seznam obrázků, tabulek a grafů .................................................................................................................... 12 1. Úvod ........................................................................................................................................................ 13 2. Přenašeči ................................................................................................................................................. 14 Klíště obecné Ixodes ricinus ........................................................................................................................ 14 3. Borrelia burgdorferi ................................................................................................................................ 19 Morfologie Borrelia burgdorferi ................................................................................................................. 21 Genom Borrelia burgdorferi ........................................................................................................................ 22 Přenos a patogenita Borrelia burgdorferi ................................................................................................... 23 4. Lymeská borelióza ................................................................................................................................... 25 Prevence nákazy a léčba lymské boreliózy ................................................................................................. 25 Diagnostika lymské boreliózy ...................................................................................................................... 27 Jak si stojí Česká republika ve výskytu lymské boreliózy? .......................................................................... 29 5. Cíle práce ................................................................................................................................................. 31 6. Metodika ................................................................................................................................................. 32 Izolace DNA z klíšťat .................................................................................................................................... 32 qPCR ............................................................................................................................................................ 33 První nested PCR ......................................................................................................................................... 34 Druhá nested PCR ....................................................................................................................................... 34 Gelová elektroforéza ................................................................................................................................... 35 Přečištění naamplikované DNA pomocí ExoSap ......................................................................................... 36 Sekvenační reakce ....................................................................................................................................... 37 Přečištění DNA pomocí EDTA a ethanolu .................................................................................................... 37 Sekvenační analýza ..................................................................................................................................... 38 7. Výsledky .................................................................................................................................................. 39 Vyhodnocení qPCR ...................................................................................................................................... 39 Vyhodnocení gelové elektroforézy ............................................................................................................. 42 Výsledky DNA sekvenování ......................................................................................................................... 42 Výskyt jednotlivých druhů Borrelia na českém území ................................................................................ 43 Fylogenetický strom .................................................................................................................................... 46 8. Diskuze .................................................................................................................................................... 47 9. Závěr ........................................................................................................................................................ 51 10. Příloha ................................................................................................................................................. 52 11. Zdroje .................................................................................................................................................. 54 Seznam použitých zkratek ACA acrodermatitis chronica atrophicans Bbsl Borrelia burgdorferi sensu lato Bbss Borrelia burgdorferi sensu stricto Bdr Borrelia direct repeat BSK Barbourův-Stoenerův-Kellyho agar BosR stress related geny CNS centrální nervová soustava EM erythema migrans IgG imunoglobulin G IgM imunoglobulin M LB lymská borelióza MEP hlavní extracelulární protein Osp Outer surface proteins PerR peroxid related geny Seznam obrázků, tabulek a grafů Obrázek 1: Morfologie klíštěte (upraveno dle: (Stafford, 2007) ..................................................................... 16 Obrázek 2: Životní cyklus klíštěte (upraveno dle: Stafford, 2007) .................................................................. 17 Obrázek 3: Buněčná stavba Bbsl (Kumar et al., 2017) ................................................................................... 21 Obrázek 4: Procentuální zastoupení pozitivních vzorků v jednotlivých krajích za rok 2017 (Zdroj: vlastní zpracování) .....................................................................................................................................................
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