Utvrđivanje prisustva Clostridium botulinum u uzorcima meda i medonosnih pčela
The investigation of the presence of Clostridium botulinum in honey and honey bee samples
Doktorand
Matović, KazimirMentor
Baltić, Milan Ž.![](/themes/MirageNardus/images/orcid.png)
Članovi komisije
Ranin, LazarKarabasil, Neđeljko
![](/themes/MirageNardus/images/orcid.png)
Mišić, Dušan
![](/themes/MirageNardus/images/orcid.png)
Nedić, Nebojša
![](/themes/MirageNardus/images/orcid.png)
Metapodaci
Prikaz svih podataka o disertacijiSažetak
U ovom radu vršeno je ispitivanje prisustva spora Clostridium botulinum u
uzorcima razlicitih vrsta meda i medonosnih pcela. Ispitano je ukupno 59 uzoraka meda
i 61 uzorak medonosnih pcela, poreklom iz razlicitih regona Republike Srbije.
Pored klasicnih mikrobioloških metoda u laboratorijskim ispitivanjima je posle
postupka dilucije, predobogacenja, centrifugovanja i membranske filtracije primenjena
metoda multipleks PCR i PCR. Odredivanje broja spora, u PCR pozitivnim uzorcima,
radeno je metodom najverovatnijeg moguceg broja (MPN).
Prisustvo spora Clostridium botulinum, metodom multipleks PCR i PCR-a,
utvrdeno je u 5 uzoraka (8,47%) meda i jednom (1,64%) uzorku medonosnih pcela. Broj
spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih
pcela 110/kg.
U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom
uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa
C.botulinum (A, E) i u jednom uzorku istovremeno tri ti...pa C. botulinum (A, B, E).
Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pcela. U
istim uzorcima meda, odnosno medonosnih pcela, klasicnim kvalitativno-kvantitativnim
metodama nije dokazano prisustvo spora Clostridium botulinum.
Detekcija spora Clostridium botulinum direktno iz neobradenih uzoraka meda i
medonosnih pcela, bez predobogacenja, nije moguca primenom metoda PCR i klasicnih
metoda mikrobiologije.
Klasicne metode mikrobiologije, ukljucujuci i metodu SRPS ISO 15213:2011,
nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske
osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je
služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim
sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna
spora/g meda.
U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih
pcela primenom metode PCR, za svaki ispitujuci uzorak moraju se izvoditi višestruka
ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne
rasporedenosti spora u uzorcima.
In this study, the presence of Clostridium botulinum spores in different types of
honey and honey bees was examined. The sum of 59 honey and 61 honey bee samples
was included in this investigation and samples originated from different regions of the
Republic of Serbia.
In addition to classical microbiological methods, after the dilution, enrichment,
spinning and membrane filtration, PCR and multiplex PCR methods were used.
Determination the number of spores in the PCR positive samples, was done using the
most likely possible number (MPN) methodology.
According to the results obtained by conventional microbiological methods and
multiplex PCR, the presence of Clostridium botulinum spores, was detected in 5 honey
samples (8.47%) and 1 (1.64%) honey bee sample. Number of spores in positive honey
samples was from 20/kg to 204/kg, and in a sample of honey bees was 110/kg.
In one honey sample, spores of type B C. botulinum were detected, in one honey
sample spores of type E, in two honey samp...les spores of types A and E, and in one
sample spores of three types of C. botulinum (A, B, E) were detected. Type E
Clostridium botulinum spores were detected in a sample of honey bees. In the same
samples of honey or honey bees, using conventional qualitative and quantitative
microbiological methods, spores of Clostridium botulinum were not detected.
The detection of Clostridium botulinum spores with PCR, multiplex PCR and
conventional microbiological methods in honey and honey bee samples is not possible
without preenrichment.
Conventional microbiological methods, including SRPS ISO 15213:2011, are
not suitable for the detection of Clostridium botulinum spores in honey samples. Due to
the low sensitivity of these methods, they can lead to false negative results. In the
honey samples which served as a positive control and wich were contaminated with
Clostridium botulinum NCTC 7272 strain, the PCR method may prove a presence of
one spore/g of honey.
In order to detect Clostridium botulinum spores in honey and honey bees
samples using PCR method, due to the small and/or unequal distribution of spores in the
samples, the test had to be repeated at least three times for each sample.
Fakultet:
Универзитет у Београду, Факултет ветеринарске медицинеDatum odbrane:
19-11-2013Projekti:
- Razvoj i standardizacija molekularnih i imunskih metoda u dijagnostici bakterijskih zoonoza (RS-MESTD-Technological Development (TD or TR)-31088)