Improved immunological membrane filter method for detection of food-borne Salmonella strains

Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Application of a DNA Hybridization–Hydrophobic-Grid Membrane Filter Method for Detection and Isolation of Verotoxigenic Escherichia coli

Todd

Szabo

Mackenzie

et al. 1999

“…For the discrimination of group N Salmonella spp., 0.1 ml of Spicer-Edwards EN antiserum complex (Difco) was stirred with 0.07 ml of HRP-protein A conjugate in 1 ml of 1% gelatin-TBS for 1 h at 40C and then increased to 40 ml with-TBS. This is a modification of the procedure described by Cerquiera-Campos et al (6).…”

Section: Methodsmentioning

confidence: 99%

“…The value of the hydrophobic grid membrane filter (HGMF) had already been demonstrated for the isolation of E. coli from food by the work of Doyle and Schoeni (8) for the detection of verocytotoxin-positive strains and by the work of Szabo et al (21) for the enumeration of E. coli 0157:H7. In addition, Cerquiera-Campos et al (6) had developed a convenient enzyme-labeled antibody (ELA) staining procedure for salmonellae which was based on the HGMF. Therefore, we developed experiments to show how effective HGMFs were for isolation and enumeration of E. coli 0157 from food with specific identification by MAbs.…”

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confidence: 99%

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods

Todd¹,

Szabo²,

Peterkin³

et al. 1988

Self Cite

An 0-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli 0157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli 0157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli 0157:H7, the organism was isolated at levels of up to 103/g. The lower limit of sensitivity was 10 E. coli 0157 per g of meat. Specific typing for E. coli 0157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.

“…Supporting this system, HGMF-based methods that use enzyme-labeled polyclonal and monoclonal antibodies have been developed for the detection of Salmonella spp. (1), enterotoxigenic Staphylococcus aureus (19), and Escherichia coli 0157 (24) in foods. Methods that use monoclonal antibodies and DNA probes for bacterial detection are specific and do not require time-consuming biochemical testing for confirmed identification of the organism (13).…”

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confidence: 99%

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe

Peterkin

Idziak

Sharpe

1991

Self Cite

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.