Acta Univ. Agric. Silvic. Mendelianae Brun. 2010, 58(5), 417-426 | DOI: 10.11118/actaun201058050417

Detekce genů kódujících enterotoxiny Bacillus cereus pomocí PCR a jejich produktů metodou BCET-RPLA a ELISA assay

Marcela Vyletělová1, Juraj Banykó2
1 Agrovýzkum Rapotín, Výzkumníků 267, 788 13 Vikýřovice, Česká republika
2 Výzkumný ústav pro chov skotu Rapotín, Výzkumníků 267, 788 13 Vikýřovice, Česká republika

Práce byla zaměřena na srovnání výsledků pro detekci přítomnosti enterotoxinů pomocí metod BCET-RPLA a ELISA immunoassay a výsledků identifikace genů odpovědných právě za syntézu složek HBL a NHE enterotoxinů (hblC, hblD, hblA a nheA, nheB, nheC). Vedle těchto genů byly detekovány i další kódující syntézu dirrhogenních a emetického enterotoxinu (bceT, cytK-1, cytK-2, entFM a ces). Kmeny Bacillus cereus byly izolovány z kravského a kozího mléka, pasterovaného mléka, mléčných produktů během technologického zpracování a z výrobního zařízení. Přítomnost HBL enterotoxinů byla stanovena pomocí metody BCET-RPLA a přítomnost NHE enterotoxinu ELISA (BDE-VIA) testem. Pro amplifikaci DNA byl použit termální cykler. Elektroforéza amplikonů probíhala po dobu 120 min při 80 V, ethidium bromidem barvené gely byly vizualizovány UV světlem, zachyceny digitálním fotosystémem (DP-001.FDC, Vilber Lourmat) a vyhodnoceny pomocí programu BioLight. Pro amplifikaci jednotlivých genů byly použity různé sekvence primerů.
Hemolytický enterotoxin HBL byl identifikován metodu BCET-RPLA u 32 kmenů B. cereus (78 %). Přítomnost hemolytického enterotoxinu NHE byla potvrzena u všech 41 sledovaných kmenů. Pozitivní detekce genů byla zjištěna následovně: gen nheA byl identifikován u 40 kmenů, nheB a nheC (41), hblA (29), hblC (39), hblD (37), bceT (8), cytK-1 (21), cytK-2 (20), entFM (41) a ces pouze v jednom případě. Přítomnost všech tří složek, které tvoří enterotoxin HBL (L2, L1 a B) a všech tří komponent, ze kterých je složen NHE enterotoxin (NheA, NheB a NheC), je nutná pro toxickou aktivitu obou enterotoxinů. Přítomnost všech tří genů nheA, nheB a nheC byla pozitivní u 40 kmenů (98 %) a genů hblA, hblC a hblD pouze u 29 kmenů (71 %). Srovnáme-li identifikaci všech tří hbl genů (pozitivní i negativní) s pozitivními či negativními výsledky BCET-RPLA, zjistíme, že jsou shodné ve 30 případech (73 %). V případě srovnání výsledků ELISA immunoassay pro stanovení NHE enterotoxinu s výsledky PCR pro všechny tři nhe geny zjistíme, že jsou shodné ve 40 případech (98 %). Pro praxi to znamená použití obou metod pro stanovení možné přítomnosti enterotoxinů, ale použítí další jiné metody pro stanovení jejich toxické activity.

mléko, Bacillus cereus, enterotoxin, geny, PCR, BCET-RPLA, ELISA assay

Detection of coding genes for enterotoxins in Bacillus cereus by PCR and their products by BCET-RPLA and ELISA Assay

Determination of enterotoxin production, diarrhoeal and emetic gene identification was studied in 41 Bacillus cereus strains isolated from raw cows' and raw goats' milk, pasteurized milk, dairy products during technological processing and from dairy plant equipment. Presence of enterotoxins was detected by BCET-RPLA (HBL) and ELISA immunoassay (NHE). Gene identification (nheA, nheB, nheC, hblA, hblC, hblD, bceT, cytK-1, cytK-2, entFM and ces) was achieved by means of PCR. Enterotoxin HBL was detected in 32 strains, enterotoxin NHE in all 41 strains. Presence of all three genes nheA, nheB and nheC was confirmed in 40 strains and genes hblA, hblC and hblD in 29 strains. Comparison of used methods was as follow: 1) BCET-RPLA (which detects L2 component) and PCR (positive or negative all three hblA, hblC and hblD gene detection) were identical in 30 (73%); 2) ELISA (NheA) and PCR (all three nheC, nheB and nheA gene expression) were identical in 40 (98%) cases isolated strains.

Keywords: milk, Bacillus cereus, genes, enterotoxins, PCR, BCET-RPLA, ELISA assay
Grants and funding:

This work was supported by Ministry of Education project MSM2678846201 and by Ministry of Agriculture project NAZV QF 3162.

Received: June 3, 2010; Published: August 6, 2014  Show citation

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Vyletělová, M., & Banykó, J. (2010). Detection of coding genes for enterotoxins in Bacillus cereus by PCR and their products by BCET-RPLA and ELISA Assay. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 58(5), 417-426. doi: 10.11118/actaun201058050417
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