20.500.12556/DKUM-90783 Analiza uporabnosti medgenske regije 16S-23S rDNA za neposredno identifikacijo vrst ocetnokislinskih bakterij iz mikrobiot kisa in kombuče s tehnologijo sekvenciranja Illumina magistrsko delo Analysis of the Utility of 16S-23S rDNA Spacer Region for the Direct Identification of Acetic Acid Bacteria Species from the Microbiota of Vinegar and Kombucha Using Illumina Sequencing Technology V magistrskem delu smo razvili metodološki pristop za neposredno identifikacijo vrst ocetnokislinskih bakterij iz kisa in kombuče na osnovi sekvenciranja odseka medgenske regije 16S-23S rDNA s tehnologijo Illumina. Do sedaj uporabljena tarčna molekula, to je gen za 16S rRNA, je namreč v primeru ocetnokislinskih bakterij evolucijsko zelo ohranjena, zato zagotavlja zanesljivo identifikacijo le do nivoja rodu. S tem namenom smo konstruirali dva para začetnih oligonukleotidov za pomnoževanje polovice medgenske regije 16S-23S rDNA, in ju uporabili za konstrukcijo knjižnic in sekvenciranje s tehnologijo Illumina. Z obema paroma začetnih oligonukleotidov smo najprej analizirali relativno abundanco vrst ocetnokislinskih bakterij v laboratorijsko pripravljenih bakterijskih združbah. Par začetnih oligonukleotidov, ki je omogočil boljšo oceno vrstne sestave bakterijske združbe, smo nadalje uporabili za analizo mikrobiot jabolčnega, bezgovega in vinskega kisa ter napitka kombuča. Preiskani vzorci so bili proizvedeni s tradicionalnim statičnim in submerznim postopkom. Postavljena metoda je uporabna za hitro identifikacijo vrst ocetnokislinskih bakterij iz substratov, kjer te bakterije predstavljajo prevladujoči del bakterijske združbe. In the master's thesis, we developed a methodological approach for the direct identification of acetic acid bacteria species from the microbiota of vinegar and kombucha based on sequencing 16S-23S rDNA internal transcribed spacer, using Illumina technology. The previously used target molecule, the 16S rRNA gene, is evolutionarily highly conserved in acetic acid bacteria, thus providing reliable identification only up to the genus level. For this purpose, we constructed two pairs of primers to amplify half of the 16S-23S rDNA internal transcribed spacer, which were then used to construct libraries and perform sequencing using Illumina technology. These primer pairs were used to analyze the relative abundance of acetic acid bacteria species in laboratory-prepared bacterial communities. The primer pair that provided a more accurate estimation of the species composition was subsequently used to analyze microbiota of apple, elderflower, and wine vinegars, and kombucha beverage. The examined samples were produced by traditional static and submerged methods. The established method is suitable for the rapid identification of acetic acid bacteria species from substrates where these bacteria represent the dominant part of the bacterial community. ocetnokislinske bakterije medgenska regija 16S-23S rDNA Illumina kis kombuča acetic acid bacteria 16S-23S rDNA internal transcribed spacer Illumina vinegar kombucha true true false [A. Ribič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-09-23 10:41:32 2024-10-08 09:35:46 2024-10-09 03:12:18 0000-00-00 00:00:00 2024 0 Maribor 0 Maribor VII, 57 f. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 579.66(043.2) 210585859 MAG_Ribic_Alja_2024.pdf MAG_Ribic_Alja_2024.pdf 1 8FA44717E7DD722BFA940C3C6BD90133 fbb2b8b6cc603ed9f936bc14a87bbb8efdbf2849287477855df9d1424321646c eb0b3af2-798a-11ef-8363-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=185706 Fakulteta za naravoslovje in matematiko 0 0 0