Zbornik - Društvo genetičara Srbije

I-Pos-12 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 33
Subotica, 30. novembar - 4. decembar 2004.
MOLEKULARNA KARAKTERIZACIJA BAKTERIJA
MLEÈNE KISELINE (BMK) IZ KOZIJEG SIRA
Milica Nikoliæ, Amarela Terziæ-Vidojeviæ, B. Jovèiæ, Lj. Topisiroviæ i Nataša Goliæ
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd
Klasiène metode za identifikaciju i determinaciju mikroorganizama, zasnovane na
kultivaciji i biohemijskim metodama, nisu uvek bile pogodne za dobijanje informacija o
mikrobiološkom sastavu razlièitih uzoraka. Analiza mikroflore klasiènim metodama je
ogranièena na mikroorganizme koji se lako kultivišu, tako da se ne dobija informacija o
kompletnoj populaciji iz uzorka. U ovom radu su korišæene molekularne tehnike kojima
je moguæe okarakterisati razlièite bakteije mleène kiseline (BMK) iz kozijeg sira. Totalna
DNK iz sira je izolovana i korišæena za PCR reakciju, gde su kao prajmeri korišæeni
U968-GC i L1401, komplementarni V6 i V8 regionima eubakterijske 16s rDNK. Sintetisani
DNK fragmenati velièine 450 bp predstavljaju zbir umnoenih DNK fragmenata
koji su poreklom od razlièitih vrsta bakterija iz sira. Fragmenti su uklonirani u plazmid
pUCEry19, a ligacionom smešom je transformisan soj E. coli DH5. Transformanti su
analizirani na prisustvo plazmida sa ukloniranim PCR fragmentom. U daljem radu biæe
uraðena DGGE analiza kloniranih fragmenata. Fingerprint tehnikom, kao što je DGGE,
se utvrðuje genetièki diverzitet mikroflore sira. Meðusobno razlièiti fragmenti æe biti
sekvenciirani u cilju precizne identifikacije mikroorganizama prisutnih u siru. Dobijeni
rezultati biæe poreðeni sa rezultatima klasiène mikrobiološke analize.
MOLECULAR CHARACTERIZATION OF LACTIC ACID
BACTERIA IN THE GOAT CHEESE
Conventional methods for the identification and determination of microorganisms, based
on cultivation and biochemical methods, usually were suitable enough for getting the information
about the composition of the microorganisms present in variable environmental
samples. However, the analysis of microflora by conventional methods is limited to
easy cultivable microorganisms and does not give the information about the complete
population in the sample. In this study molecular techniques were used to characterize
different lactic acid bacteria (LAB) from a goat cheese. The first step was the isolation of
total DNA from the cheese that has been used as a template for PCR reaction. PCR, by
using primers U968-GC and L1401 complementary to the V6 and V8 region of
eubacterial 16s rDNA was performed. Obtained DNA products of 450 bp represent the
sum of amplified DNA fragments originated from different species of bacteria from the
cheese. These fragments were cloned into a plasmid pUCEry19, and ligation mixture was
transformed into a competent E. coli strain DH5. Transformants were screened for the
presence of plasmids containing cloned PCR fragments. The aim of further research will
be the DGGE analysis of the cloned fragments. DGGE, as a fingerprinting technique is
suitable to describe the genetic diversity of the cheese composition. Different fragments
will be sequenced and the obtained data would be used for the identification of the microorganisms
present in the goat cheese. These results will be compared with results obtained
by classical microbiological methods.