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master's thesis
Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.)
Zagreb: University of Zagreb, Faculty of Science, 2010. urn:nbn:hr:217:348442
Haluška, Ivana
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

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Haluška, I. (2010). Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.) (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:348442

Haluška, Ivana. "Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.)." Master's thesis, University of Zagreb, Faculty of Science, 2010. https://urn.nsk.hr/urn:nbn:hr:217:348442

Haluška, Ivana. "Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.)." Master's thesis, University of Zagreb, Faculty of Science, 2010. https://urn.nsk.hr/urn:nbn:hr:217:348442

Haluška, I. (2010). 'Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.)', Master's thesis, University of Zagreb, Faculty of Science, accessed 22 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:348442

Haluška I. Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.) [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2010 [cited 2024 November 22] Available at: https://urn.nsk.hr/urn:nbn:hr:217:348442

I. Haluška, "Usporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.)", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2010. Available at: https://urn.nsk.hr/urn:nbn:hr:217:348442

Metadata
TitleUsporedba metoda izolacije DNA u detekciji fitoplazmi ribosomskih skupina 16SrV i 16SrXII-A iz vinove loze (Vitis vinifera L.)
Title (english)Comparison of DNA isolation methods in detection of 16SrV and 16SrXII-A ribosomal group phytoplasmas from grapevine (Vitis vinifera L.)
AuthorIvana Haluška
MentorMartina Šeruga Musić (mentor)
Committee memberMartina Šeruga Musić (predsjednik povjerenstva)
Committee memberIvana Ivančić Baće (član povjerenstva)
Committee memberDubravka Hranilović (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2010, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Abstract
Fitoplazme (Candidatus 'Phytoplasma') su bakterije bez stanične stijenke iz razreda Mollicutes čiji se životni ciklus sastoji od replikacije u biljkama i kukcima. Uzročnici su nekoliko stotina bolesti na različitim biljkama, uključujući važne poljoprivredne vrste. Budući da ih nije moguće uzgajati in vitro, njihova klasifikacija predstavlja problem, no razvojem molekularnih metoda, poput ugniježđene lančane reakcije polimerazom (PCR) i lančane reakcije polimerazom u stvarnom vremenu (real-time PCR), omogućena je njihova detekcija i identifikacija. Ukupna genomska DNA biljke može se izolirati različitim metodama pa je cilj ovog rada bio usporediti osjetljivost detekcije fitoplazmi ugniježđenim i real-time PCR-om, ovisno o metodi izolacije DNA. Ukupne nukleinske kiseline 6 uzoraka fitoplazmama zaražene vinove loze (Vitis vinifera L.) izolirane su kratkom CTAB-metodom za izolaciju TNA i komercijalnim kompletima OmniPrep™ for Plant i DNeasy Plant Mini Kit. Prisutnost fitoplazmi dokazana je umnažanjem gena za 16S rRNA ugniježđenim PCR-om, te gena map metodom real-time PCR. Kako bi se ispitala osjetljivost detekcije, korištene su različite koncentracije kalupa izolirane DNA. Kod 5 analiziranih uzoraka dokazana je prisutnost fitoplazmi ribosomske skupine 16SrXII-A dok je kod jednog uzorka detektirana fitoplazma skupine 16SrV. Usporedba rezultata ukazala je da se izoliranjem ukupne DNA kratkom CTAB-metodom kod većine analiziranih uzoraka dobiva najveća osjetljivost detekcije fitoplazmi pomoću obje korištene metode.
Abstract (english)
Phytoplasmas (Candidatus 'Phytoplasma') are wall-less bacteria from the class Mollicutes. Their life cycle involves replication in plants and insects. They cause several hundred diseases in various plants, including important agricultural species. Since phytoplasmas cannot be cultivated in vitro, their classification is problematic, but with development of molecular methods, such as nested and real-time polymerase chain reaction (PCR), phytoplasma detection and identification was made possible. Total genomic DNA can be isolated from the plant by various methods.The aim of this thesis was to compare sensitivity of phytoplasma detection by nested and real-time PCR, depending on DNA isolation method. Total nucleic acids of 6 phytoplasma infected grapevine (Vitis vinifera L.) samples were isolated by short CTAB method for TNA isolation, and by commercial kits OmniPrep™ for Plant and DNeasy Plant Mini Kit. Presence of phytoplasmas was detected by nested PCR amplifying 16S rRNA gene and real-time PCR amplifying map gene. Different concentrations of DNA template were used in order to compare the sensitivity of detection. In 5 of the analyzed samples presence of phytoplasmas belonging to 16SrXII-A ribosomal group was detected, while in one sample we detected 16SrV group phytoplasma. Comparison of the results indicated that DNA isolation using short CTAB method gave the highest sensitivity of phytoplasma detection in most of the analyzed samples by both nested and real-time PCR.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:348442
Study programmeTitle: Biology; specializations in: Molecular biology
Course: Molecular biology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra biologije (magistar/magistra biologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2018-03-23 11:58:54