Comparison of a microtitration plate ELISA with a standard cultural procedure for the detection of Salmonella spp. in chicken.

A rapid antibody-capture enzyme-linked immunosorbent assay (ELISA) detecting a wide range of Salmonella serotypes and employing only one culture stage was used to analyze the giblets and body cavity rinsings from frozen chickens. The results from the ELISA were compared with those obtained using a standard cultural procedure in current use in two laboratories, Norwich (N) and Ipswich (I), of the Public Health Laboratory Service (PHLS) in the UK. ELISAs were carried out on the same samples at each of two PHLS laboratories and at the Institute of Food Research with good agreement (94% and 90%). When compared with the cultural method there was 80% and 70% agreement with the ELISA with the PHLS(N) and PHLS(I) samples. The ELISA appeared to have a false-positive rate of 17% (samples from PHLS(N)) but on reculture of the "negative" samples this rate fell to 7%. The false-negative rate for the ELISA was 26% (samples from PHLS(N)) which appeared to be due to insufficient growth of the Salmonella spp. in the single cultural step employed in the ELISA rather than lack of recognition by the antibodies. The problem of false negatives with the cultural method is also discussed. These results are comparable to previously published studies relating immunoassays and the conventional procedure for Salmonella detection when analyzing similar samples. Suggestions are made as to how further increases in ELISA efficiency might be brought about.     

Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp.

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.     

Comparison of four different methods for Salmonella detection in fecal samples of porcine origin

Performances of four detection methods were evaluated for recovery of Salmonella spp. in naturally contaminated fecal specimens of porcine origin. The NMKL 71 method consisted of enrichment in Rappaport-Vassiliadis broth and plating on xylose-lysine-desoxycholate medium, whereas the SP-VG-M002 method relied on a Diasalm enrichment followed by streaking on xylose-lysine-tergitol 4 agar (XLT-4). The VIDAS SLM method was composed of double enrichment in Muller-Kauffmann tetrathionate broth and in M broths before processing in a VIDAS device. If the results were positive, the VIDAS ICS immunoenrichment was performed and the result transferred onto three different selective media. The VIDAS ICS protocol is an immunoconcentration step followed by plating on XLT-4. Seventy-eight samples were tested with all four methods simultaneously, leading to 34 positive samples with at least one method. For this assay, VIDAS SLM revealed 31 positive samples (91.2%), whereas the average positive percentage of the three other methods was 37.3% (P < 0.001). Two-paired comparisons with the VIDAS SLM method were also performed. McNemar values were systematically highly significant (P < 0.001). The proportion of agreement was significantly inferior (P < 0.05) for the comparison of VIDAS ICS and VIDAS SLM (68.7%) compared with the two other paired comparisons (average percentage, 81.5%). The conclusion reached by this trial is that VIDAS SLM significantly improves the recovery of Salmonella in naturally contaminated fecal specimens. For the paired-comparisons, NMKL 71 and SP-VG-M002 were comparable in terms of efficiency, whereas the VIDAS ICS protocol, as established by the manufacturer for food samples only, seemed less efficient than the other two.     

Evaluation of a 5'-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5'-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with "gold standard" methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5'-nuclease assay was able to identify the organism in 100% of the repetitions when 10(2) CFU/ml or more organisms were present in pure cultures and 10(3) CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10(5) CFU/ml or more organisms were present in pure culture and 10(6) CFU/g or more organisms were present in inoculated ground pork. The 5'-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5'-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5'-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

Comparison of a real-time PCR method with a culture method for the detection of Salmonella enterica serotype enteritidis in naturally contaminated environmental samples from integrated poultry houses

Conventional culture methods have traditionally been considered the "gold standard" for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis-specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis-specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.     

Evaluation of culture enrichment procedures for use with Salmonella detection immunoassay

To design efficient culture strategies for use with immunoassays to detect Salmonella in food, the growth of these organisms was investigated according to the Bacteriological Analytical Manual (BAM) and enrichment-immunoassay (EI) culture procedures. The cultures were further evaluated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The BAM procedure includes pre-enrichment in nutrient broth (NB) for 16 h followed by selective enrichment in either Rappaport-Vassiliadis (RV) or tetrathionate brilliant green (TBG) broth for 16 h. The EI procedure includes pre-enrichment in NB for 4 h, selective enrichment in RV for 16 h and post-enrichment in NB for 4 h. The effects of different incubation times for pre- and post-enrichment, and different culture media for selective enrichment (TBG and RV) and post-enrichment in NB and Brain Heart Infusion broth (BHI) on the growth of the bacteria and ELISA titers in the EI procedure were also investigated. Salmonella enteritidis and S. typhimurium inoculated at different initial concentrations between 0.1 and 35 CFU/ml grew to similar concentrations of 10(7) to 10(8) colony forming unit (CFU)/ml in pure culture and generally 2 to 4 fold lower concentrations (P<0.05) in mixed culture using spiked chicken rinse. In the BAM procedure, the concentration of Salmonella cultured in RV was higher (P<0.01) than that in TBG. The cultures in TBG showed positive results for ELISA, but those in RV were generally negative. In the EI procedure, the ELISA titers from cultures post-enriched in NB or BHI were higher (P<0.01) when TBG, as compared to RV, was used for selective enrichment. Post-enrichment in BHI yielded higher numbers of Salmonella and higher ELISA titers than those in NB (P<0.05) for post-enrichment. This study demonstrated that in both culture procedures small numbers of Salmonella could be increased to at least 10(7) CFU/ml which is detectable by most ELISAs, and that the type of the culture media used may have a significant impact on ELISA results.     

An optimised HS-SPME-GC-MS method for the detection of volatile nitrosamines in meat samples

A method to detect volatile nitrosamines in meat samples was developed using headspace sampling by solid-phase microextraction (HS-SPME), with analysis by GC-MS. A 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane fused silica fibre was selected to extract a total of nine volatile nitrosamines: N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosomorpholine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosodi-n-butylamine, and N-nitrosodiphenylamine. Extraction at 65°C for 45 min with 36% (w/v) NaCl were the optimal conditions determined for the extraction of nine nitrosamines. Excellent linearity was obtained for all analytes with determination coefficients greater than 0.997. Recovery rates were between 92 and 113%. The relative standard deviation ranged from 0.81 to 8.0% for six of the nine compounds, and from 16 to 32% for the other three. For seven out of nine nitrosamines, limits of detection were below 3.6 µg kg^?1 and the limits of quantification were below 12 µg kg^?1. The nitrosamine levels in four varieties of processed meat products were investigated to assess the applicability of the method. Based on the results, the developed HS-SPME-GC-MS method proved to be a simple and efficient technique to detect seven out of nine nitrosamines in meat products with adequate sensitivity, accuracy and precision.     

Comparison between two commercial ELISAs and a culture procedure for the detection of Listeria spp.

 The efficiency of two commercial enzyme-linked immunosorbent assays (ELISAs), the Oxoid Listeria rapid test and the Tecra Listeria visual immunoassay for the detection of Listeria spp. was compared with that of a culture method. Of the 60 samples examined by ELISA and the culture procedure, Listeria was detected and confirmed by culture in 20, 34 and 44 samples by the Tecra, Oxoid and microbiological methods, respectively. The overall sensitivity of the Tecra and Oxoid tests was 50% and 93%, respectively. Both ELISAs had a specificity of 100%. The efficiency of the Oxoid test was 95%, while that of the Tecra assay was 67%. Differences in the results of the Oxoid test and those of the culture method were not statistically significant; however, the differences between the results of the Tecra assay and the culture procedure were significant. The Oxoid test was less labour intensive, less time consuming and easier to perform than the Tecra assay. It was concluded that the Oxoid test is a good alternative method to the culture procedures when screening for the presence of Listeria spp. in foods, especially when a large number of samples have to be analysed. Received: 24 June 1997     

Comparison between two commercial ELISAs and a culture procedure for the detection of Listeria spp.

 The efficiency of two commercial enzyme-linked immunosorbent assays (ELISAs), the Oxoid Listeria rapid test and the Tecra Listeria visual immunoassay for the detection of Listeria spp. was compared with that of a culture method. Of the 60 samples examined by ELISA and the culture procedure, Listeria was detected and confirmed by culture in 20, 34 and 44 samples by the Tecra, Oxoid and microbiological methods, respectively. The overall sensitivity of the Tecra and Oxoid tests was 50% and 93%, respectively. Both ELISAs had a specificity of 100%. The efficiency of the Oxoid test was 95%, while that of the Tecra assay was 67%. Differences in the results of the Oxoid test and those of the culture method were not statistically significant; however, the differences between the results of the Tecra assay and the culture procedure were significant. The Oxoid test was less labour intensive, less time consuming and easier to perform than the Tecra assay. It was concluded that the Oxoid test is a good alternative method to the culture procedures when screening for the presence of Listeria spp. in foods, especially when a large number of samples have to be analysed. Received: 24 June 1997     

Impedance detection of Salmonella in processed animal protein and meat.

The impedance technique showed a detection rate (95%) equal to that of conventional enrichment for raw meat contaminated with Salmonella. For processed animal protein samples impedance was less sensitive. A commercially available Easter and Gibson impedance medium used for the selective enrichment of salmonellae proved superior to the laboratory prepared equivalent for the detection of Salmonella in processed animal protein. The rate of false-positive results with the impedance technique was high.     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

Comparison between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork carcass sponge samples

VIDAS Salmonella (VIDAS-SLM) is an automated system that uses the enzyme-linked fluorescent assay method to detect Salmonella species. This study evaluated the efficacy of the VIDAS-SLM method in detecting Salmonella species in pork carcass sponge samples gathered from 10 slaughter plants in Taiwan. Two hundred fifty-seven pork carcass sponge samples were screened by the VIDAS-SLM method and by the culture method in parallel. While 18 sponge samples were found to test positive by both methods, the VIDAS-SLM method detected four additional positive samples for which the culture method failed to recover Salmonella. The specificity of the VIDAS-SLM method was found to be 0.98, and its sensitivity was 1.0, since no false-negative results occurred. Artificially inoculated Salmonella at concentrations as low as 5.0 x 10(0) CFU/ml was detected in the heat-inactivated sponge sample in the presence or absence of 5.0 x 10(4) CFU of Citrobacter freundii per ml. Thus, the VIDAS-SLM method is a rapid screening method and a potential alternative to the time- and labor-intensive culture method.     

Development of a fimY-based loop-mediated isothermal amplification assay for detection of Salmonella in food

In this study, we developed a rapid and sensitive fimY-based loop-mediated isothermal amplification (LAMP) assay on a real-time turbidimeter platform for the detection of Salmonella in food. Since turbidity of the reaction mixture would increase in correlation with the DNA yield, real-time monitoring of the LAMP reaction was achieved by real-time turbidimeter. Time threshold values which indicate positive results for 81 Salmonella strains of different serotypes ranged from 36 to 40 min. For the 20 non-Salmonella strains, turbidity did not increase in the reaction mixture. When testing 10-fold serial dilutions of Salmonella Typhimurium-ATCC 14128 DNA by LAMP, the time threshold ranged from 36 to 52 min on the real-time turbidimeter. The detection limit was 13 cells per reaction in pure culture, up to 10-fold more sensitive than that of PCR. When applied in deli food samples, the LAMP assay was able to detect Salmonella even though the sample was contaminated with very low concentration after 3 h enrichment culture. Increase in turbidity was observed on real-time turbidimeter. Additionally, the LAMP results detected by naked-eye or turbidity consistently matched with each other. Results from this study showed that the fimY-based LAMP assay is an effective method for the rapid detection of Salmonella.     

Evaluation of an immunomagnetic separation-real-time PCR assay for the rapid detection of Salmonella in meat

The aim of this study was the comparison of an immunomagnetic separation (IMS)-real-time PCR assay for the detection of Salmonella with the cultural reference method according to and 35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS-real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe-based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS-real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS-real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance index kappa, which defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS-real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index kappa had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS-real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.     

Comparison of a new enrichment procedure for Shiga toxin-producing Escherichia coli with five standard methods

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g(-1). Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 x 10(8) CFU ml(-1) and 1.80 x 10(6) CFU ml(-1) after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.     

A comparison of standard cultural methods for the detection of foodborne Salmonella.

The sensitivity of the standard cultural method of the International Organization for Standardization (ISO 6579 and ISO 3565 combined) was compared to that of the Health Protection Branch (HPB) procedure for the detection of foodborne Salmonella. Of 195 foods tested, 84 (43.1%) were found to contain salmonellae by one or more cultural conditions. Of these, 75 (89.3%) and 68 (81.0%) were identified by the ISO and HPB methods, respectively. The apparent lack of agreement between both methods likely stemmed from the low indigenous numbers of salmonellae in several food homogenates, and unequal transfer of the target microorganism into homologous ISO and HPB pre-enrichment broths. The sensitivities of the commercially available Muller-Kauffmann tetrathionate broth (MKTBG43, Oxoid CM343), and a closely-related medium prepared with Oxoid CM29 tetrathionate base varied from 86.9 to 89.3%, and were deemed equivalent to that obtained with the ISO formulation of MKTBG43 (89.3%). Comparatively fewer contaminated samples were identified from selenite cystine (SC35) and selenite brilliant green (SBG35) enrichment cultures (82.1-83.3%). The high selectivity and saccharide-independent response of the bismuth sulfite agar medium warrants its consideration as a mandatory plating medium in ISO methodologies for the effective detection of typical and atypical biotypes of foodborne Salmonella spp.     

Comparison of various assays used for detection of beta-lactam antibiotics in poultry meat

In this study, microbiological tests for the detection of beta-lactam antibiotics in meat and meat products were evaluated. The traditional FPT (four plate test, containing Bacillus subtilis and Kocuria rhizophila), BsDA (Bacillus stearothermophilus disc assay) and a newly developed microbiological test, Premi®Test (containing Bacillus stearothermophilus) were included in the study. The limit of detection (LOD) of the Premi®Test was compared with the LOD of the traditional methods. The detection limits of the tests were determined by using beta-lactam antibiotic standards dissolved in meat juice, as well as meat tissue obtained from laying hens after experimental administration of amoxicillin. Positive samples, based on inhibition of growth of the organism in the test, were confirmed by high performance liquid chromatography (HPLC). Growth inhibition in the traditional tests is visible as a clear zone on the plate, whereas for Premi®Test, this is based on the absence of a colour change of the test. The LODs of antibiotics tested were as follows: Penicillin G (PENG) 5 µg kg^-1, amoxicillin (AMOX) 10 µg kg^-1, ampicillin (AMP) 25 µg kg^-1, oxacillin (OXA) 30 µg kg^-1, and cloxacillin (CLOX) 30 µg kg^-1 on the plate with Bacillus stearothermophilus. Beta-lactam antibiotics can be detected also on one plate seeded with Kocuria rhizophila, although the LODs are higher: PENG 10 µg kg^-1, AMOX 25 µg kg^-1, AMP 30 µg kg^-1, OXA 50 µg kg^-1, and CLOX 50 µg kg^-1. Premi®Test was performed according to the Standard Operating Procedure intended for detection of beta-lactam antibiotics in poultry tissues with following LODs: PENG 4 µg kg^-1, AMOX 5 µg kg^-1, AMP 5 µg kg^-1, OXA 40 µg kg^-1, CLOX 50 µg kg^-1. All tests are able to detect beta-lactam antibiotics such as penicillin G, ampicillin, amoxicillin, oxacillin and cloxacillin below the maximum residue level (MRL). However, the detection limits of the Premi®Test for PENG, AMOX and AMP were below the limits of BsDA and the plate containing Kocuria rhizophila.     

Comparison of immunohistochemical,histochemical and immunochemical methods for the detection of wheat protein allergens in meat samples and cooked,dry, raw and fermented sausage samples

Nowadays is it a common practice to add vegetable protein in the production of meat products. Because of the possible substitution of high-quality raw meat with vegetable protein without the labelling the product package and because of the allergenic potential of many vegetable proteins, it is important to develop accurate methods for its detection. The objective of the study was to compare histochemical, immunochemical (ELISA, ALERT gliadin screening test) and immunohistochemical methods for the detection of wheat protein in meat samples and sausages. Histochemical methods were useful for the detection of flour in meat samples, but the immunohistochemical method was better for the detection of wheat protein. ALERT gliadin screening test detected gliadin from 10?mg?kg^?1, while an immunohistochemical method detected wheat protein concentrations from 1?g?kg^?1 and an ELISA method detected wheat protein concentrations from 4?g?kg^?1. ALERT gliadin screening test showed results within 1 day, whilst an ELISA detection method took 2 days, and an immunohistochemical procedure took 5 days at the soonest, all including sample preparation. This study also focused on optimisation of an immunohistochemical method for samples of cooked sausage. In addition, three samples were sufficient for wheat protein detection at a concentration of 1?g?kg^?1 (and greater) with a confidence level greater than 95%.     

Bacteriophage-based enrichment coupled to immunochromatographic strip-based detection for the determination of Salmonella in meat and poultry

Immunochemical-based methods for the detection of Salmonella in food can be complicated by the presence of closely related, immunocrossreactive non-Salmonella species in the sample that may cause false-positive results. To circumvent this problem, specific bacteriophages against immunocrossreactive, non-Salmonella bacteria were used in the sample enrichment step to suppress their growth and improve the performance of an immunochromatographic strip-based detection method for Salmonella. Cross-reactive bacteria were isolated from various food sources and were characterized with a panel of Salmonella somatic O antigen-specific monoclonal antibodies. These cross-reactive bacteria were primarily Citrobacter spp. and Escherichia coli with serology shared with Salmonella serogroups B, D, and F. These bacteria were used as hosts for the isolation of specific lytic bacteriophages. When formulated with the primary enrichment, the bacteriophage cocktail significantly reduced false positives with a broadly reactive immunochromatographic test strip. This was demonstrated in both artificially and naturally contaminated meat. False positives in naturally contaminated beef samples were reduced from 32 of 115 samples tested to zero. In raw meat and poultry with a relatively high bioburden (>10(5) CFU/g), the use of the bacteriophage-based enrichment procedure gave improved recovery of Salmonella compared with the conventional culture-based reference method. This was observed when coupled to either test strip-based or selective agar-based detection. The use of specific bacteriophages for the control of immunocrossreactive and competitive microflora during the food sample enrichment step provides a new approach for enhancing the performance of both immunological- and cultural-based detection methods.