Analýza mikroflóry v sýrech pomocí DGGE
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Molekulárno-biologické metódy sú rýchlou a účinnou pomôckou pri identifikácii mikroorganizmov v reálnych vzorkách. Cieľom tejto diplomovej práce bola analýza mikroflóry v kontrolných a kontaminovaných nezrejúcich syroch. DNA izolovaná z analyzovaných vzoriek bola použitá pre optimalizáciu PCR s primérmi s GC svorkou pre rozdelenie amplikónov pomocou DGGE. Produkty DGGE boli po optimalizácii reamplifikované a pripravené pre sekvenáciu. DNA izolovaná z analyzovaných vzoriek bola použitá pre PCR v reálnom čase s analýzou kriviek topenia amplikónov s vysokým rozlíšením (HRMA). Pomocou DGGE a HRMA analýzy boli porovnané amplikóny syrov s bakteriálnymi kultúrami izolovanými kultivačne z týchto syrov. Porovnaním polohy amplikónov bolo zistené, že kontaminujúcimi mohli byť Bacillus licheniformis, Staphylococcus epidermidis a Acinetobacter baumanii/calcoaceticus. Analýzou sekvencií amplikónov syrov a nálevov bola zistená, prítomnosť Bacillus sp. a ďalších mikroorganizmov radených do piatich rodov. Z nich boli vykultivovaní zástupcovia troch rodov z Preto sa predpokladá kontaminácia Bacillus sp., alebo mikroorganizmy, ktoré neboli použitými metódami kultivovateľné. Metóda je vhodná pre analýzu komplexnej mikroflóry v syroch a nálevoch po ďalšej optimalizácii.
Molecular biological methods are fast and efficient tool for the identification of microorganisms in real samples. The aim of this diploma thesis was analysis of microflora in control and contaminated brined cheeses. DNA isolated from analyzed samples was used to optimize the PCR course using primers with GC clamp on the distribution of amplicons using DGGE. DGGE products were reamplified after optimization and prepared for DNA sequencing. DNA isolated from analyzed samples was used in real-time PCR with high resolution melt analysis of the amplicons (HRMA). Samples of cheese and bacterial cultures isolated from cheeses were compared by DGGE and HRMA. Comparing the position of the amplicons was found that contaminants may be Bacillus licheniformis, Staphylococcus epidermidis, and Acinetobacter baumanii/ calcoaceticus. Sequence analysis of cheese and pickles amplicons, the presence of Bacillus sp. and other microorganisms spree in five genera were detected. Representatives of the tree genera were cultured. It is considered contamination Bacillus sp., or microorganisms which are not culturable methods used. The method is suitable for the analysis of complex microflora in cheese and pickles after further optimization.
Molecular biological methods are fast and efficient tool for the identification of microorganisms in real samples. The aim of this diploma thesis was analysis of microflora in control and contaminated brined cheeses. DNA isolated from analyzed samples was used to optimize the PCR course using primers with GC clamp on the distribution of amplicons using DGGE. DGGE products were reamplified after optimization and prepared for DNA sequencing. DNA isolated from analyzed samples was used in real-time PCR with high resolution melt analysis of the amplicons (HRMA). Samples of cheese and bacterial cultures isolated from cheeses were compared by DGGE and HRMA. Comparing the position of the amplicons was found that contaminants may be Bacillus licheniformis, Staphylococcus epidermidis, and Acinetobacter baumanii/ calcoaceticus. Sequence analysis of cheese and pickles amplicons, the presence of Bacillus sp. and other microorganisms spree in five genera were detected. Representatives of the tree genera were cultured. It is considered contamination Bacillus sp., or microorganisms which are not culturable methods used. The method is suitable for the analysis of complex microflora in cheese and pickles after further optimization.
Description
Citation
ČAKAJDOVÁ, M. Analýza mikroflóry v sýrech pomocí DGGE [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2015.
Document type
Document version
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Language of document
cs
Study field
Potravinářská chemie a biotechnologie
Comittee
prof. RNDr. Ivana Márová, CSc. (předseda)
prof. Ing. Peter Šimko, DrSc. (místopředseda)
doc. Ing. Jiřina Omelková, CSc. (člen)
doc. RNDr. Alena Španová, CSc. (člen)
doc. Ing. Bohuslav Rittich, CSc. (člen)
doc. Ing. Pavel Diviš, Ph.D. (člen)
Date of acceptance
2015-06-02
Defence
1. Diplomantka seznámila členy komise s náplní a cílem diplomové práce.
2. Byly přečteny posudky na diplomovou práci.
3. Diplomantka akceptovala všechny připomínky oponenta a na všechny otázky odpověděla v plné šíři.
Diskuse
prof. Márová: Veškerá DNA byla vyizolována přímo ze sýra ?
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení