Use of an Elevated Temperature and Novobiocin in a Modified Enzyme-linked Immunosorbant Assay for the Improved Recovery of Salmonella from Foods

A modified colorimetric enzyme-linked immunosorbant assay (ELISA) for Salmonella detection was compared to the standard culture method of the Bacteriological Analytical Manual/Association of Official Analytical Chemists (BAM/AOAC) using 20 artificially contaminated foods (1,200 test samples). The modifications to the current methodology consisted of an elevated incubation temperature of 42°C for the tetrathionate selective broth and M-broth postenrichments, as well as addition of 10 μg/ml novobiocin to the M-broth. The microtiter plate as not agitated during assay incubation, and centrifugation steps were eliminated from the protocol. This modified ELISA method was at least as productive as the standard AOAC culture method for the food samples tested. No false-positive reactions were encountered. The false-negative incidence was 1.5% for the immunoassay and 5.3% by the AOAC cultural method. The incidence of agreement between the methods was 96.7%.

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Elevated-Temperature, Colorimetric, Monoclonal, Enzyme-Linked Immunosorbent Assay for Rapid Screening of Salmonella in Foods: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/77.2.374 ◽

1994 ◽

Vol 77 (2) ◽

pp. 374-394 ◽

Cited By ~ 7

Author(s):

Karl F Eckner ◽

Wendy A Dustman ◽

Michael S Curiale ◽

Russell S Flowers ◽

Barbara J Robison

Keyword(s):

Elevated Temperature ◽

Immunosorbent Assay ◽

Collaborative Study ◽

False Negative ◽

Culture Method ◽

Soy Flour ◽

Rapid Screening ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa Method ◽

Milk Chocolate

Abstract A collaborative study was performed by 30 laboratories in 3 sets of trials to validate a modified colorimetric monoclonal enzyme-linked immunosorbent assay (ELISA) method for Salmonella detection. The modifications to the current methodology included incubation of enrichments and postenrichments at an elevated temperature, addition of novobiocin to the M-broth post-enrichment, and elimination of the centrifugation and agitation steps. Five artificially contaminated foods (nonfat dry milk, milk chocolate, dried egg, ground black pepper, and soy flour) and 1 naturally contaminated food (raw ground turkey) were analyzed. The artificially contaminated foods were inoculated with individual Salmonella serotypes at a high (10–50 cells/25 g) and low (1–5 cells/25 g) contamination level. Results from the modified ELISA method were compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method. In 2 of the food products, milk chocolate and pepper, a number of laboratories isolated Salmonella from uninoculated control samples, thus invalidating their data. As a result, there were too few laboratories remaining with valid data, and these foods were repeated. In the completed study, there were 11 false negative results obtained by the modified ELISA method, while there were 28 false negatives produced by the BAM/AOAC procedure. There were 11 ELISA positive assays which could not be confirmed by culture methods. Statistically, there were no differences between the modified, colorimetric, monoclonal ELISA and the reference culture method in all foods except raw turkey, where the ELISA method was more productive. The colorimetric monoclonal enzyme immunoassay (Salmonella-Tek) method for detecting Salmonella in all foods has been adopted first action by AOAC INTERNATIONAL.

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Comparison of Two Enzyme Immunoassays for Recovery of Salmonella spp. from Four Low-Moisture Foods

Journal of Food Protection ◽

10.4315/0362-028x-55.8.601 ◽

1992 ◽

Vol 55 (8) ◽

pp. 601-604 ◽

Cited By ~ 7

Author(s):

GERALDINE ALLEN JUNE ◽

PATRICIA S. SHERROD ◽

WALLACE H. ANDREWS

Keyword(s):

False Negative ◽

Culture Method ◽

Lower Number ◽

Salmonella Spp ◽

False Negatives ◽

Enzyme Immunoassays ◽

Assay Result ◽

Standard Culture ◽

Low Moisture Foods ◽

Number Of Cells

Two enzyme immunoassays (Salmonella-Tek™ and Report™) were compared with the standard culture method of the Association of Official Analytical Chemists (AOAC) and the Food and Drug Administration's Bacteriological Analytical Manual (BAM) for the recovery of Salmonella spp. from four low-moisture foods. Two protocols were used to compare the effectiveness of the two immunoassays: i) foods were contaminated in the dry state; or ii) serial tenfold dilutions of Salmonella spp. were inoculated into the postenrichments after incubation. Of three hundred 25-g test portions inoculated in the dry state, 199 gave confirmed positive reactions with the Salmonella-Tek™ assay, 193 with the Report™ assay, and 206 with the AOAC/BAM method. There were seven false-negative reactions with Salmonella-Tek™ and 13 false negatives with the Report™, a false negative being defined as one that was negative by the enzyme immunoassay but was confirmed positive by the AOAC/BAM culture method. When the postenrichments were inoculated after incubation, a lower number of cells gave a positive assay result with the Salmonella-Tek™ system than with the Report™ system, indicating greater sensitivity

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VIDAS® Enzyme-Linked Immunofluorescent Assay for Detection of Listeria in Foods: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/83.4.903 ◽

2000 ◽

Vol 83 (4) ◽

pp. 903-918 ◽

Cited By ~ 15

Author(s):

Vidhya Gangar ◽

Michael S Curiale ◽

Armando D’Onorio ◽

Ann Schultz ◽

Ronald L Johnson ◽

...

Keyword(s):

False Positive Rate ◽

Collaborative Study ◽

False Negative ◽

Traditional Culture ◽

Culture Method ◽

Contamination Level ◽

Culture Methods ◽

Positive Rate ◽

Standard Culture ◽

Lis Method

Abstract The VIDAS LIS method and the traditional culture methods for detection of Listeria species in food were evaluated in a multilaboratory comparative study. The 6 foods tested were either naturally contaminated or inoculated with 3 different concentrations of Listeria. Results for each food and each contamination level with the VIDAS LIS method were as good as or better than those obtained with the traditional culture method. Of 1558 samples tested, 935 were positive: 839 by the VIDAS method and 809 by standard culture methods. Overall false negative rates were 10.3 and 13.5% for the VIDAS LIS and culture methods, respectively. The false positive rate for the VIDAS LIS assay was 1.4% based on 9 VIDAS LIS positive assays that did not confirm positive by isolation of Listeria. The agreement between the VIDAS LIS and culture methods for all samples tested was 86%.

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Comparison of Salmonella Bio-EnzaBead Immunoassay Method and Conventional Culture Procedure for Detection of Salmonella in Foods

Journal of Food Protection ◽

10.4315/0362-028x-50.5.379 ◽

1987 ◽

Vol 50 (5) ◽

pp. 379-385 ◽

Cited By ~ 18

Author(s):

KARL F. ECKNER ◽

RUSSELL S. FLOWERS ◽

BARBARA J. ROBISON ◽

JEROME A. MATTINGLY ◽

DAMIEN A. GABIS ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Enzyme Immunoassay ◽

Culture Method ◽

Food Samples ◽

Selective Enrichment ◽

Conventional Culture ◽

Significant Difference ◽

Standard Culture ◽

Raw Shrimp ◽

Culture Procedure

A rapid enzyme immunoassay screening procedure (EIA) utilizing two monoclonal antibodies specific for salmonellac was compared to the standard culture method (BAM/AOAC) on 1,289 samples representing 26 food types. The samples consisted of 760 artificially inoculated, 150 naturally contaminated, and 379 uninoculated food samples. There were 594 samples positive by the EIA (optical densities greater than 0.2 at 405 nm), of which 568 were confirmed culturally from M-broth. A total of 570 samples was positive by the BAM/AOAC procedure. Of the foods tested, there was no significant difference between the two methods, with the exception of cake mix and raw shrimp. The EIA was significantly better for detecting Salmonella in cake mix, while the culture procedure was more productive for shrimp. The method employed a 24 ± 2-h preen-richment, an 18-h selective enrichment, and a 6-h M-broth post-enrichment. The EIA assay required an additional 2 h for a total of 48 h, compared to a minimum of 4 d by BAM/AOAC.

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Comparison of Colorimetric Monoclonal Enzyme Immunoassay Screening Methods for Detection of Salmonella in Foods

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/73.1.43 ◽

1990 ◽

Vol 73 (1) ◽

pp. 43-50

Author(s):

Michael S Curiale ◽

Mary Joan Klatt ◽

Barbara J Robison ◽

Lisa T Beck

Keyword(s):

Enzyme Immunoassay ◽

Magnetic Beads ◽

False Negative ◽

Screening Method ◽

False Negative Rate ◽

Culture Method ◽

Food Samples ◽

Assay Method ◽

Screening Methods ◽

Well Assay

Abstract A colorimetric enzyme immunoassay (EIA) method for detection of Salmonella in foods has been compared to the AOAC colorimetric monoclonal EIA screening method (986.35,15th ed.; 46.B21-46.B29, 14th ed.). The assays use the same monoclonal antibodies and have similar reactivity toward Salmonella. However, the new assay uses antibody-coated microtiter wells instead of coated magnetic beads to capture Salmonella antigens. Compared with the bead assay, the coated-well assay format requires significantly less time to complete, and was consistently able to detect lower levels of Salmonella In mixed culture. Compared to the standard AOAC culture method for food samples, the plate assay was as productive. No false negatives were obtained by the immunoassay; the false negative rate was 1.1% by the culture method. The rate of agreement between the 2 methods was 99.1 %. The official final action bead assay method for Salmonella in foods, 986.35, and the same assay for use with low-moisture foods, 987.11, have been modified official first action to use antibody-coated microtiter strip-wells

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Evaluation of TaqMan PCR Assay for Detecting Salmonella in Raw Meat and Shrimp

Journal of Food Protection ◽

10.4315/0362-028x-62.4.329 ◽

1999 ◽

Vol 62 (4) ◽

pp. 329-335 ◽

Cited By ~ 55

Author(s):

BON KIMURA ◽

SUSUMU KAWASAKI ◽

TATEO FUJII ◽

JUN KUSUNOKI ◽

TAKESHI ITOH ◽

...

Keyword(s):

False Negative ◽

Culture Method ◽

Nuclease Activity ◽

Food Samples ◽

Pcr Assay ◽

Culture Methods ◽

Conventional Culture ◽

Fluorogenic Probe ◽

Taqman Pcr ◽

Polymerase Chain

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.

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Stability of Deoxynivalenol in Heat-Treated Foods†

Journal of Food Protection ◽

10.4315/0362-028x-62.8.962 ◽

1999 ◽

Vol 62 (8) ◽

pp. 962-964 ◽

Cited By ~ 49

Author(s):

CHARLENE E. WOLF-HALL ◽

MILFORD A. HANNA ◽

LLOYD B. BULLERMAN

Keyword(s):

High Temperature ◽

Extraction Method ◽

Elisa Method ◽

Immunosorbant Assay ◽

Dog Food ◽

High Temperature And Pressure ◽

Heat Treated ◽

Enzyme Linked Immunosorbant Assay ◽

Temperature And Pressure

The effects of high-temperature and -pressure processing of foods spiked with deoxynivalenol (DON) were examined. In extruded corn grits, extruded dry dog food, and autoclaved moist dog food, there were no significant reductions (P < 0.05) in DON after processing. Autoclaved cream-style corn showed a reduction in DON of only 12%. Overall, DON was stabile to the high temperature and pressure processes tested. The use of an α-amylase in the extraction method for analysis by an enzyme-linked immunosorbant assay (ELISA) improved the recovery of DON from the spiked extruded and autoclaved products by as much as 26% over the standard ELISA method.

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Validated PCR Assay for the Routine Detection of Salmonella in Food

Journal of Food Protection ◽

10.4315/0362-028x-69.2.282 ◽

2006 ◽

Vol 69 (2) ◽

pp. 282-287 ◽

Cited By ~ 19

Author(s):

N. S. BANSAL ◽

V. GRAY ◽

F. McDONELL

Keyword(s):

False Negative ◽

Food Samples ◽

Bacterial Cells ◽

Pcr Assay ◽

Culture Methods ◽

Perfect Agreement ◽

Selective Enrichment ◽

Negative Results ◽

Standard Culture ◽

Pure Cultures

We developed a rapid and reliable PCR assay with genus-specific primers for the detection of Salmonella in food samples. With these primers, no primer-specific amplicons were detected when challenged with cultures of microorganisms other than salmonellae, and positive results, i.e., Salmonella-specific bands, were obtained with pure cultures of all 125 Salmonella isolates tested, which represented 100 serovars. The PCR assay was optimized using both pure cultures and artificially inoculated food samples. The assay results were compared with those of the Australian standard culture methods, using more than 500 “naturally” contaminated food samples, over a period of 9 years. Food samples were subjected to nonselective preenrichment in buffered peptone water followed by selective enrichment in Rappaport Vassiliadis (RV) broth and mannitol selenite cystine (MSC) broth. A simple sample preparation method was developed based on concentrating bacterial cells from 1 ml of RV or MSC broths. The PCR results were in perfect agreement with the results of the standard culture methods; no false-positive or false-negative results were obtained. However, the PCR assay was extremely rapid, and results could be obtained within 4 h of testing of enrichment broths.

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