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Primena seroloških metoda, multipleks lančane reakcije polimeraze i sekvenciranja gena za 16S ribozomalnu RNK i identifikaciji serovarijeteta vrste Salmonella enterica podvrste enterica

Application of serotyping, multiplex polymerase chain reaction and sequencing of the gene for 16S ribosomal RNA in identification of serovars of Salmonella enterica subspecies enterica

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2019
Disertacija.pdf (2.333Mb)
IzvestajKomisije22209.pdf (406.5Kb)
Author
Kiškarolj, Ferenc
Mentor
Mišić, Dušan
Committee members
Šenerović, Lidija
Radojičić, Sonja
Radojičić, Marina
Milanov, Dubravka
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Abstract
U ovoj doktorskoj disertaciji je ispitana mogućnost primene simpleks i multipleks PCR protokola sa prethodno opisanim prajmerima za preciznu identifikaciju najčešće izolovanih serovarijeteta Salmonella vrsta na teritoriji Vojvodine. Takođe je ispitana efikasnost primene metode sekvenciranja gena za 16Ѕ rRNK u preciznoj identifikaciji salmonela. Ispitano je 107 izolata Salmonella enterica ssp. enterica identifikovanih klasičnom serotipizacijom kao: Infantis (52), Enteritidis (33), Tenessee (5), Mbandaka (4), Montevideo (3), Havana (2), Lille (2), Senftenberg (2), Typhimurium (1), Agona (1), Derby (1) i Livingstone (1). Korišćena je tripleks PCR reakcija u detekciji bcfC, steB i sdf lokusa, optimizovana tokom preliminarnih ispitivanja. Primenom tripleks PCR, samo S. Enteritidis je mogla precizno da bude razlikovana od drugih serovarijeteta, dok su ostali serovarijeteti na osnovu rezultata tripleks PCR svrstavani u dve grupe (Grupa 1 i Grupa2). S. Infantis, najzastupljeniji serovarijetet ...među ispitanim izolatima nije mogao biti precizno identifikovan jer se primenom tripleks PCR nije razlikovao od ostalih članova Grupe 2. Za njegovu konačnu identifikaciju je primenjen simpleks PCR za umnožavanje segmenta fljB koji je karakterističan samo za S. Infantis. Sekvenciranje gena za 16Ѕ rRNK je primenjeno u identifikaciji izolata devet različitih serovarijeteta. Poredjenjem rezultata dobijenih u klasičnoj serotipizaciji i u PCR, uočeno je slaganje u 91 slučaju od ukupno 107 (85%). Od 33 izolata S. Enteritidis njih 31 (94%) je dao očekivan PCR profil. U slučaju S. Infantis PCR je potvrdila identifikaciju u samo 75% (39 od 52) slučajeva. Ostali ispitani serovarijeteti dali su očekivane PCR profile. Sekvenciranje gena za 16Ѕ rRNK je bilo pouzdano samo u određivanju pripadnosti ispitanih izolata rodu Salmonella.

Aim of this doctoral thesis was to test the applicability of published primers for the identification of serovars isolated most frequently in the north part of our country using multiplex and simplex PCR reactions. The efficacy of sequencing of gene for 16S ribosomal RNA for identification of Salmonella strains was also checked. The study was conducted on 107 serotyped Salmonella enterica ssp. enterica isolates. Strains belonged to the following serovars: Infantis (52), Enteritidis (33), Tenessee (5), Mbandaka (4), Montevideo (3), Havana (2), Lille (2), Senftenberg (2), Typhimurium (1), Agona (1), Derby (1) i Livingstone (1). A triplex PCR protocol (bcfC, steB, sdf) optimized during preliminary investigations was used. This reaction separated S. Enteritidis from other strains, while the remaining serovars divided in two distinct groups (Group 1 and Group 2). As the used PCR protocol could not differentiate S. Infantis, the most numerous serovar among isolates, from other members of the... Group 2, its identification was completed using a simplex PCR reaction targeting fljB specific for S. Infantis. Comparison of the results of serotyping and PCR protocols revealed a concordance in 91 cases from the total of 107 (85%). From 33 strains serotyped as S. Enteritidis 31 (94%) had the predicted PCR profile. Regarding S. Infantis, identification based on genomic markers confirmed this serovar only in 75% of cases (39 from 52). Other serovars showed their typical PCR profiles. Sequencing of gene for 16S ribosomal RNA could reliably determine only that tested isolates are the members of the genus Salmonella

Faculty:
Универзитет у Београду, Факултет ветеринарске медицине
Date:
28-10-2019
Keywords:
Salmonela enterica / Salmonela enterica / serovar / serotyping / multiplex PCR / sequencing of gene for 16S rRNA / serovarijetet / serotipizacija / multipleks PCR / sekvenciranje gena za 16S rRNK
[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_nardus_12117
URI
http://eteze.bg.ac.rs/application/showtheses?thesesId=7293
https://nardus.mpn.gov.rs/handle/123456789/12117
https://fedorabg.bg.ac.rs/fedora/get/o:21070/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=51827727

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