Cloth-based hybridization array system for the detection of Clostridium botulinum type A, B, E, and F neurotoxin genes.

A simple cloth-based hybridization array system was developed for the characterization of Clostridium botulinum isolates based on the botulinum neurotoxin serotype. Bacterial isolates were subjected to a multiplex PCR incorporating digoxigenin-dUTP and primers targeting the four botulinum neurotoxin gene serotypes (A, B, E, and F) predominantly involved in human illness, followed by hybridization of the amplicons with an array of toxin gene-specific oligonucleotide probes immobilized on polyester cloth and subsequent immunoenzymatic assay of the bound digoxigenin label. This system provided sensitive and specific detection of the different botulinum neurotoxin gene markers in a variety of C. botulinum strains, exhibiting the expected patterns of reactivity with a panel of target and nontarget organisms.

Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA.

AIMS: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin-based enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens. METHODS AND RESULTS: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high-affinity adsorbent for E. coli O157 LPS antigens, which were immunoenzymatically detected using anti-E. coli O157 antibody-enzyme conjugates. With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red and QuantaBlu produced only slight improvement in the performance characteristics of the polymyxin-ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB). On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos compared with the chromogenic substrate p-nitrophenylphosphate. CONCLUSIONS: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E. coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB. SIGNIFICANCE AND IMPACT OF THE STUDY: The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods.

Cloth-based hybridization array system for the detection of multiple antibiotic resistance genes in Salmonella enterica subsp. enterica serotype Typhimurium DT104.

AIMS: A simple DNA macroarray system was developed for detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. METHODS AND RESULTS: A multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used to simultaneously amplify seven marker sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. This system provided sensitive detection of the different genetic markers in the S. Typhimurium DT104 genome, giving positive reactions with as few as 10 CFU, and the hybridizations were highly specific, with no reactions of amplicons with heterologous probes on the array. CONCLUSIONS: This cloth-based hybridization array system (CHAS) provides a simple, cost-effective tool for monitoring S. Typhimurium DT104 in foods and their production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The CHAS is a simple and cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the detection and characterization of food pathogens.

Detection of hazelnut proteins in foods by enzyme immunoassay using egg yolk antibodies.

An enzyme immunoassay (EIA) was developed for the detection of hazelnut proteins in foods. This assay used inexpensive chicken egg yolk antibodies in a sandwich EIA format for the immunospecific capture and detection of hazelnut proteins present in a variety of different food matrices. The assay was able to detect less than 1 ppm of hazelnut protein in most of the foods tested and did not exhibit any appreciable cross-reactivity with other nuts or food matrices. This assay will be a useful tool for the food industry and regulatory agencies that wish to test foods for the presence of undeclared hazelnut allergens.

Swab-based enzyme immunoassay system for detection of meat residues on food contact surfaces as a hygiene monitoring tool.

An enzyme immunoassay system based on the use of a macroporous swab as a solid phase for the capture and subsequent immunoenzymatic detection of immunoglobulin G (IgG) from meat residues on food contact surfaces was developed as a hygiene-monitoring tool. Moistened polyester swabs coated with anti-bovine or anti-chicken IgG were rubbed on the test surface, and the captured IgG was subsequently detected directly on the swabs by brief sequential reactions with anti-bovine or anti-chicken IgG-peroxidase conjugate and chromogenic peroxidase substrate.

Performance of the Dynabeads anti-Salmonella system in the detection of Salmonella species in foods, animal feeds, and environmental samples.

The immunomagnetic separation Dynabeads anti-Salmonella technique was evaluated for the detection of Salmonella species in a variety of foods, feeds, and environmental samples. Salmonella cells in preenrichment broths were captured using the Dynabeads anti-Salmonella system and were then washed and plated on indicator media. A total of 308 naturally contaminated samples were analyzed, including 46 cheese and egg products, 183 animal feeds, and 79 environmental swabs. The results of the Dynabeads method gave 100% correlation with the results of the standard culture technique used for foods and the modified semisolid Rappaport-Vassiliadis method used for feeds and environmental samples.

Polymacron enzyme immunoassay system for detection of naturally contaminating Salmonella in foods, feeds, and environmental samples.

A simple dot blot enzyme immunoassay was developed to screen enrichment broth cultures for the presence of Salmonella. This unique system utilizes macroporous polyester cloth (Polymacron) with an inexpensive hemoglobin coating to provide a high-affinity adsorbent for lipopolysaccharide (LPS) antigens in test samples. Bound LPS antigens are then detected using a monoclonal antibody conjugate recognizing a core oligosaccharide epitope common to all salmonellae frequently found in foods and related samples. The entire test (not including enrichment culture) could be completed in less than 1 h. The performance of this assay was evaluated in the analysis of enrichment broth cultures from a variety of egg and dairy products, chicken carcasses, animal feeds, and food-processing plant environmental samples for the presence of Salmonella.

Specificity of monoclonal antibodies to Campylobacter jejuni lipopolysaccharide antigens.

Monoclonal antibodies (Mabs) were produced to the lipopolysaccharide antigens of Campylobacter jejuni strain 1249 (Penner serotype O:2/63). A polymyxin-cloth based enzyme immunoassay (pCEIA) was used for initial screening and for evaluating the specificity of these antibodies. Seven Mabs reacted with at least 11 and as many as 14 of 15 C. jejuni strains (representing 8 different Penner serotypes). These seven Mabs did not cross-react with any of 16 non-Campylobacter bacteria commonly encountered in food, with only two exceptions. Several combinations of these Mabs in pairs reacted with all 15 C. jejuni strains. These results suggest that pCEIA employing two of these Mabs in combination is potentially useful for detection of Campylobacter jejuni in foods and other samples.

Assay of Salmonella in enrichment cultures of foods, feeds and environmental samples by the polymyxin-cloth enzyme immunoassay.

A variety of foods, animal feeds and environmental samples were analyzed for the presence of Salmonella using the polymyxin-cloth enzyme immunoassay (p-CEIA) system. Salmonella lipopolysaccharide (LPS) antigens were captured from test samples on polymyxin-coated polyester cloth, followed by immunoenzymatic detection of bound antigens using a monoclonal antibody recognizing an LPS common core oligosaccharide. Dairy and egg products, animal feeds and environmental samples from food processing plants were pre-enriched for 24 h, followed by selective enrichment for a further 24 h in either tetrathionate brilliant green (TBG), selenite cystine (SC) or brain-heart infusion broth containing 0.5% yeast extract, 0.5% cholate and 0.3% selenite (BYCS). The samples were assayed by the p-CEIA after each stage of enrichment. After selective enrichment, the p-CEIA gave results which were in complete agreement with the standard culture technique in the analysis of all foods examined. On the other hand, a combination of selective enrichment and the p-CEIA out-performed the Modified Semi-Solid Rappaport Vassiliadis (MSRV) method in screening pre-enrichment cultures of feeds and environmental samples. Use of the new selective medium BYCS prior to performing the p-CEIA gave the highest recovery of Salmonella from feeds and environmental samples.

Effect of temperature and agitation on enrichment of Escherichia coli O157:H7 in ground beef using modified EC broth with novobiocin.

The effects of temperature and agitation on the enrichment of Escherichia coli O157:H7 in meat using modified EC broth with novobiocin (mEC + n) were studied. Enrichment at 37 degrees C was compared to 42 degrees C, both with and without shaking. Incubation at 42 degrees C without shaking effectively suppressed ground beef microflora while allowing good growth of E. coli O157:H7 cells. Cells inoculated into ground meats (beef, pork, turkey) were readily detected by enrichment for 24 h in mEC + n at 42 degrees C without shaking, followed by screening the enrichment cultures using a rapid and inexpensive commercially available enzyme immunoassay system, the E. coli O157 Rapitest.

Microtitre plate hybridization system for detection of thermophilic Campylobacter rRNA.

A microtitre plate nucleic acid probe hybridization system was developed for the detection of ribosomal RNA from thermophilic Campylobacter (Camp. jejuni, Camp. coli, Camp. lari and Camp. upsaliensis). A specific DNA probe obtained by amplification of 23S rRNA sequences using the polymerase chain reaction technique was immobilized on a microtitre plate, and used for hybridization with target 23S rRNA from cell lysates. The RNA-DNA hybrids thus formed in the wells were detected by an immunoenzymatic assay using a monoclonal antiRNA-DNA hybrid antibody. The sensitivity of this system was 2.7 x 10(4) cells ml(-1). This simple, sensitive and inexpensive hybridization and immunoenzymatic assay system should facilitate the detection of Campylobacter in food and clinical samples.

A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences.

A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth.

Detection of Campylobacter jejuni in food and poultry viscera using immunomagnetic separation and microtitre hybridization.

Thermophillic Campylobacter and Camp. jejuni were detected from samples of chicken liver, gall bladder, muscle and contaminated milk and chicken meat after an enrichment step by using immunomagnetic capture of cells with monoclonal antibody against a specific outer membrane protein of thermophilic Campylobacter. The detection of captured cells was achieved using two different hybridization methods. In one of the methods, the captured cells were lysed by guanidine isothiocyanate and the 23S rRNA was reacted with a microtitre plate-immobilized rDNA probe specific for thermophilic Campylobacter. In the other method, the captured cells were subjected to lysis by ultrasonication and the genomic DNA reacted with a microtitre plate-immobilized RNA probe specific for Camp.jejuni. Detection of the RNA-DNA hybrids formed in the wells was carried out using a monoclonal anti-RNA-DNA hybrid antibody.

Microtitre plate riboprobe system for detection of ultrasonicated Campylobacter jejuni genomic DNA.

Microtitre plate nucleic acid probe hybridization systems were developed for the detection of thermophilic Campylobacter and Campylobacter jejuni. Specific RNA probes obtained by in vitro transcription of DNA templates synthesized by polymerase chain reaction using two sets of specific primers incorporating bacteriophage T7 promoter sequences were immobilized on a microtitre plate. The hybridizations were carried out on samples of genomic DNA sheared by ultrasonication. Optimum conditions for the ultrasonic treatment were determined in order to obtain the highest degree of hybridization with immobilized RNA probe. Finally, detection of RNA-DNA hybrids in the wells was accomplished by an immunoenzymatic assay using a monoclonal anti-RNA-DNA hybrid antibody. This rapid, simple hybridization and immunoenzymatic assay system will facilitate the detection of Campylobacter in foods and clinical samples.

Application of cloth-based enzyme immunoassay for the characterization of monoclonal antibodies to Salmonella lipopolysaccharide antigens.

The specificity, detection limit, and stability of twelve anti-Salmonella monoclonal antibodies (Mabs) were evaluated by cloth-based enzyme immunoassay (CEIA) and polymyxin-cloth based enzyme immunoassay (p-CEIA). Using the p-CEIA, five Mabs were found to react with cholate extracted lipopolysaccharide (LPS) antigens of all 44 Salmonella strains representing 19 different serogroups examined, with the exception of the one strain of serogroup-O tested. These five Mabs did not react with cholate extracts of any of 16 Gram-positive or Gram-negative non-Salmonella bacteria tested. The detection limit of purified S. typhimurium LPS antigen in the p-CEIA was approximately 40 ng for four of the Mabs and approximately 20 ng for the other Mab. Four of the five Mabs were stable during storage at 22 degrees C-23 degrees C for 24 h. These four Mabs are potentially useful for the immunodetection of Salmonella in foods and other samples.

Salmonella detection by the polymyxin-cloth enzyme immunoassay using polyclonal and monoclonal detector antibodies.

Several commercially available O-antigen polyclonal antisera and a monoclonal antibody to the core region of lipopolysaccharide (LPS) were examined as sources of detector antibodies in a polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) for Salmonella. In this assay, polymyxin-coated polyester cloth captured the LPS antigens from Salmonella broth cultures, followed by immunoenzymatic detection of the captured LPS using specific antibodies. Pools of polyvalent antisera reacted with all of the Salmonella strains tested, but also gave cross-reactions with some non-Salmonella bacteria. On the other hand, the monoclonal antibody gave positive reactions with all of the Salmonella tested except serogroup O-strains, but did not react with any of the non-Salmonella bacteria. The monoclonal antibody supplemented with a single factor serogroup O:35 rabbit antiserum was able to detect the serogroup O-strains without causing any cross-reactions with the non-Salmonella bacteria. As an example of the applicability of this assay system, low levels of Salmonella cells spiked into various food samples were successfully detected after an overnight enrichment in broth.

Multiplex riboprobes for the detection of virulent Yersinia enterocolitica and simple methods for their preparation.

A simple multiplex riboprobe system for detection of virulent Yersinia enterocolitica was developed using a pool of RNA probes specific for various chromosomal and plasmid-borne virulence gene sequences (yadA, virC, ail and yst). Riboprobes were synthesized by a rapid, simple and efficient technique involving in vitro transcription of polymerase chain reaction-generated templates incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. After dot blotting target DNA samples on nitrocellulose and hybridization with the riboprobes, the RNA: DNA hybrids formed were detected by a simple immunoenzymic assay involving sequential reactions with an anti-RNA : DNA hybrid monoclonal antibody, anti-mouse Ig-peroxidase conjugate and chromogenic or chemiluminescent enzyme substrate solution. This multiplex riboprobe system targeting both chromosomal and plasma-borne sequences permitted detection of virulent Y. enterocolitica, regardless of plasmid loss during handling of cultures, and was unreactive with a virulent Y. enterocolitica, other Yersinia and other bacteria. This system resulted in a significant improvement in the limit of detection in comparison to that obtained with individual probes.

Rapid and economical detection of Salmonella enteritidis in eggs by the polymyxin-cloth enzyme immunoassay.

A rapid, simple and economical procedure for the detection of Salmonella enteritidis in eggs was developed. The contents of whole eggs inoculated with low numbers of S. enteritidis were mixed with a minimal volume of a nutrient-rich broth (1:2 ratio of egg to broth) and incubated overnight. The lipopolysaccharide (LPS) antigens of S. enteritidis were extracted by heating in the presence of cholate. The antigens were captured on polymyxin-coated polyester cloth, and the captured antigens were detected by sequential reactions with anti-serogroup D1 rabbit antiserum, anti-rabbit antibody-peroxidase conjugate and tetramethylbenzidine substrate solution. This polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) was highly specific for salmonellae bearing the factor O:9 antigen, reacting in the assay of 19 S. enteritidis strains tested, including two rough isolates, but not with salmonellae lacking the factor O:9 antigen or non-Salmonella bacteria. The threshold sensitivity of the polymyxin-CEIA for S. enteritidis suspensions was ca. 10(6) cfu/ml. This combined enrichment culture and polymyxin-CEIA required less than 24 h to complete and detected as few as 1-2 S. enteritidis cfu inoculated into a whole egg. This procedure should facilitate the routine monitoring of S. enteritidis in large numbers of egg samples.

Transcriptional enhancement of the Listeria monocytogenes PCR and simple immunoenzymatic assay of the product using anti-RNA:DNA antibodies.

A method was developed to enhance the sensitivity of a Listeria monocytogenes PCR detection system by in vitro transcription of amplicons incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. The resulting transcript can be detected by hybridization with a DNA probe immobilized in the wells of a microtiter plate, followed by immunoenzymatic assay of the RNA-DNA hybrids with an anti-RNA-DNA hybrid antibody. This highly sensitive method was reactive in the assay of various L. monocytogenes isolates but not with other Listeria or non-Listeria species.

Use of inexpensive O antisera as the detecting antibodies for Salmonella antigens in the polymyxin-cloth enzyme immunoassay.

The application of O antigen-specific antisera to the detection of Salmonella lipopolysaccharide (LPS) antigens was examined in an enzyme immunoassay using polymyxin-coated polyester cloth. The LPS antigens were extracted with deoxycholate and captured on polymyxin-cloth. A mixture of rabbit antisera to Salmonella O antigens was allowed to react with the captured antigens, and the reacted antibodies were detected by an anti-rabbit IgG-peroxidase conjugate. The assay gave positive results with 40 different Salmonella serotypes which represented more than 99.9% of the serogroups isolated from over 122,000 food, feed and environmental samples analysed at Laboratory Services Division, Agriculture Canada, from 1975 to 1990. Strong cross-reactivity with Staphylococcus aureus was eliminated by pregrowth of the organisms in the presence of sodium deoxycholate. The O antisera were commercially available and are more economical than monoclonal or affinity purified polyclonal antibodies.