Comparison of DNA isolation methods in detection of 16SrV and 16SrXII-A ribosomal group phytoplasmas from grapevine (Vitis vinifera L.)

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.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