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2011, vol. 61, br. 2-3, str. 239-245
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Poboljšana senzitivnost i reproducibilnost PCR metode za detekciju Listeria spp. i L. monocytogenes u mleku
Improved sensitivity and reproducibility of the PCR method for detection of Listeria spp. and L. monocytogenes in milk
aInstitut za higijenu i tehnologiju mesa, Beograd, Srbija bUniverzitet u Beogradu, Institut za molekularnu genetiku i genetičko inženjerstvo, Srbija
e-adresa: nalebeli@eunet.rs
Projekat: Izučavanje gena i molekularnih mehanizama u osnovi probiotičke aktivnosti bakterija mlečne kiseline izolovanih sa područja zapadnog Balkana (MPNTR - 173019)
Sažetak
Listeria monocytogenes, prouzrokovač listerioze kod ljudi i životinja, je fakultativan intraćelijski mikroorganizam široko rasprostranjen u prirodi. U cilju izolacije i detekcije L. monocytogenes iz hrane koriste se tradicionalne mikrobiološke i nove molekularno-genetičke metode. Cilj ovog rada je bio povećanje osetljivosti i ponovljivosti PCR metode u detekciji L. monocytogenes u mleku. U tu svrhu, uzorci pasterizovanog mleka su kontaminirani serijskim razblaženjima sojeva L. monocytogenes 4b ATCC 119115 i Listeria innocua ATCC 33090. Dobijeni rezultati na veštački kontaminiranim uzorcima pasterizovanog mleka, ukazuju da osetljivost PCR metode zavisi od perioda inkubacije i izbora prajmera. Najbolji rezultati su dobijeni nakon 24 h inkubacije, pomoću prajmera za hlyA gen, kada je bilo moguće detektovati 1 ćeliju L. monocytogenes tj. 1 CFU/ml.
Abstract
Listeria monocytogenes is a facultative intracellular Grampositive bacterium, ubiquitous in nature and capable of causing listeriosis in humans and animals. Conventional microbiological techniques and modern molecular approaches are currently used for the isolation and detection of L. monocytogenes in food samples. The aim of this study was to improve the sensitivity and reproducibility of PCR for the detection of Listeria spp. in milk. For that purpose milk samples were artificially inoculated with serial dilutions of L. monocytogenes 4b ATCC 19115 and L. innocua ATCC 33090. The results obtained on artificially contaminated milk samples indicated that incubation time and target genes have an influence on the sensitivity of PCR detection. The best results were obtained after 24 h of preenrichment, with primers complementary to the hlyA gene, when it was possible to detect 1 CFU/mL of Listeria spp.
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