Validation of the ANSR®E. coli O157:H7 Method for Detection of E. coli O157:H7

2016 ◽  
Vol 99 (3) ◽  
pp. 705-716 ◽  
Author(s):  
Ryan Viator ◽  
Susan Alles ◽  
Quynh-Nhi Le ◽  
Edan Hosking ◽  
Evan Meister ◽  
...  
Keyword(s):  
Target Genes ◽  
Ground Beef ◽  
Reference Method ◽  
Single Step ◽  
Fluorescence Detector ◽  
Method Performance ◽  
E Coli ◽  
Reference Methods ◽  
Inspection Service ◽  
The U.S

Abstract A performance validation of the ANSR® for E. coli O157:H7 method was conducted in selected food matrixes. This assay uses selective nicking enzyme amplification technology to amplify target genes. Samples are enriched for 12–24 h and then lysed. The assay is completed within 40 min using real-time detection in a combination incubator/fluorescence detector and software. When 44 distinct strains of Escherichia coli O157:H7 and 6 strains of E. coli O157:NM were tested for inclusivity, all 50 strains produced positive results. In exclusivity testing, 57 strains representing 33 species of closely related Gram-negative bacteria belonging to the Enterobacteriaceae family, including 11 non-H7 O157 strains and shiga toxin-producing E. coli other than O157:H7, were evaluated. All 57 nontarget strains generated negative ANSR assay results. Using 80% lean ground beef and beef trim (approximately 20% fat), ANSR method performance was compared to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure. ANSR performance with baby spinach and sprout irrigation water was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method. ANSR method performance was not statistically different to that of the reference methods using two different enrichment options. For ground beef and beef trim, the standard enrichment in modified Tryptone Soya Broth can be analyzed using the ANSR assay with a 1:10 dilution of the enrichment in phosphate-buffered saline and produces equivalent results to the reference method. Additionally, in most matrixes tested (exception is spinach which required 24 h enrichment) the assay offers great efficiency and flexibility over the reference method with a 12–24 h single-step enrichment. Equivalent results were observed at both time points (12 and 24 h) to reference methods. Small changes to the assay parameters minimally affected ANSR method performance. Finally, accelerated stability results from three independently manufactured lots support a shelf-life of 6 months when stored at 4°C.

Validation of the ANSR® for Campylobacter Method for Detection of Thermophilic Campylobacter spp. in Chicken Carcass Rinse and Turkey Sponge Samples

2016 ◽  
Vol 99 (6) ◽  
pp. 1555-1564 ◽  
Author(s):  
Ryan Viator ◽  
Susan Alles ◽  
Quynh-Nhi Le ◽  
Edan Hosking ◽  
Preetha Biswas ◽  
...  
Keyword(s):  
Target Genes ◽  
Reference Method ◽  
Common Species ◽  
Single Step ◽  
Fluorescence Detector ◽  
Method Performance ◽  
Chicken Carcass ◽  
Inspection Service ◽  
Nicking Enzyme ◽  
Performance Validation

Abstract A performance validation of the ANSR® for Campylobacter method was conducted in selected matrixes. This assay used selective nicking enzyme amplification technology to amplify target genes. Samples were enriched for 20 to 24 h and then lysed. The assay was completed within 50 min using real-time detection in a combination incubator/fluorescence detector and software. When 50 distinct strains of Campylobacter jejuni, C. lari, or C. coli were tested for inclusivity, all 50 strains produced positive results. In exclusivity testing, 31 strains of related organisms, including seven nontarget Campylobacter strains and other common species, were evaluated. All 31 species generated negative ANSR assay results, including the nontarget Campylobacter strains. The ANSR for Campylobacter method was compared to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method using naturally contaminated chicken carcass rinse or turkey carcass sponge samples. ANSR method performance was not statistically different from the reference method using two different enrichment options. Equivalent results were observed at both time points (20 and 24 h) and in both atmospheres (microaerobic and aerobic) to reference methods. Method performance with chicken carcass rinse was confirmed in an independent laboratory study. Additionally, in robustness testing, small, deliberate changes to the assay parameters minimally affected ANSR method performance. Finally, accelerated stability results from three independently manufactured lots supported a shelf life of 6 months when stored at 4°C. The ANSR assay offered greater efficiency and flexibility when compared to the reference method with a 20–24 h single-step enrichment in a microaerobic or an aerobic atmosphere.

Validation of the ANSR® Listeria monocytogenes Method for Detection of Listeria monocytogenes in Selected Food and Environmental Samples

2016 ◽  
Vol 99 (1) ◽  
pp. 112-123
Author(s):  
Oscar Caballero ◽  
Susan Alles ◽  
Quynh-Nhi Le ◽  
R Lucas Gray ◽  
Edan Hosking ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Storage Temperature ◽  
Reference Method ◽  
Single Step ◽  
Stainless Steel Surface ◽  
Method Performance ◽  
Inspection Service ◽  
Nicking Enzyme ◽  
Performance Results ◽  
The U.S

Abstract Work was conducted to validate performance of the ANSR® for Listeria monocytogenes method in selected food and environmental matrixes. This DNA-based assay involves amplification of nucleic acid via an isothermal reaction based on nicking enzyme amplification technology. Following single-step sample enrichment for 16–24 h for most matrixes, the assay is completed in 40 min using only simple instrumentation. When 50 distinct strains of L. monocytogenes were tested for inclusivity, 48 produced positive results, the exceptions being two strains confirmed by PCR to lack the assay target gene. Forty-seven nontarget strains (30 species), including multiple non-monocytogenes Listeria species as well as non-Listeria, Gram-positive bacteria, were tested, and all generated negative ANSR assay results. Performance of the ANSR method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for detection of L. monocytogenes in hot dogs, pasteurized liquid egg, and sponge samples taken from an inoculated stainless steel surface. In addition, ANSR performance was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method for detection of L. monocytogenes in Mexican-style cheese, cantaloupe, sprout irrigation water, and guacamole. With the single exception of pasteurized liquid egg at 16 h, ANSR method performance as quantified by the number of positives obtained was not statistically different from that of the reference methods. Robustness trials demonstrated that deliberate introduction of small deviations to the normal assay parameters did not affect ANSR method performance. Results of accelerated stability testing conducted using two manufactured lots of reagents predicts stability at the specified storage temperature of 4°C of more than 1 year.

scholarly journals Validation of iQ-Check E. coli O157:H7 Real-Time PCR Test Kit for Detection of Escherichia coli O157:H7 in Selected Foods

2009 ◽  
Vol 92 (4) ◽  
pp. 1095-1104 ◽  
Author(s):  
Wendy F Lauer ◽  
Sylvie Tymciu ◽  
Caroline D Sidi ◽  
Pierre Sonigo
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Reference Method ◽  
Target Sequence ◽  
Apple Cider ◽  
E Coli ◽  
Test Kit ◽  
Significant Difference ◽  
The U.S

Abstract iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

scholarly journals USDA FSIS and FDA BAM Culture Methods BBL CHROMagar Salmonella Prepared Plated and Difco Dehydrated Culture Media

2009 ◽  
Vol 92 (2) ◽  
pp. 459-470 ◽  
Author(s):  
Vicki Ritter ◽  
Nancy Dick
Keyword(s):  
Culture Media ◽  
False Positive Rate ◽  
Ground Beef ◽  
Reference Method ◽  
Chi Square ◽  
Shell Eggs ◽  
Reference Methods ◽  
Raw Fish ◽  
Chi Square Analysis ◽  
The U.S

Abstract BBL and Difco CHROMagar Salmonella (CS) was evaluated internally and externally for the recovery of Salmonella in raw chicken, raw ground beef, raw fish, lettuce, and shell eggs. The raw chicken and ground beef were processed according to the U.S. Department of Agriculture, Food Safety and Inspection Service reference methods. The raw fish, lettuce, and shell eggs were processed according to the U.S. Food and Drug Administration, Bacteriological Analytical Manual procedures. Only raw chicken was found to be naturally contaminated with Salmonella; all other matrixeswere seededwith an appropriate dilution of organism to achieve fractionally positive results. Salmonella strains were permitted to equilibrate with the culture-negative matrixes for 48 h at 4C. Twenty 25 g samples of each food matrix plus 5 uninoculated samples were processed. CS prepared plates (CS PPM) and laboratory prepared plates from dehydrated culture media (CS DCM) were evaluated with the reference method media. A total of 16 positive cultures were obtained from the raw chicken samples, 17 in the raw ground beef, 18 in the raw fish and lettuce, and 11 in the shell eggs. A Chi-square analysis was performed on each of the food matrixes. BBL CS produced comparable results with the reference methods on all matrixes, resulting in a method agreement of 100 based on the Chi-square results. In testing known isolates the sensitivity and specificity was determined to be 94. Specificity improved to 98 when tetrathionate broth enrichment was used. A negative- and false-positive rate of 6 was found with known isolates. No false negatives were found in testing the food matrixes. The performance of the CS prepared plate and laboratory prepared plate was identical.

scholarly journals Reveal 8-Hour Test System for Detection of Escherichia coli O157:H7 in Raw Ground Beef, Raw Beef Cubes, and Iceberg Lettuce Rinse: Collaborative Study

2001 ◽  
Vol 84 (3) ◽  
pp. 719-736 ◽  
Author(s):  
Charles B Bird ◽  
Rebecca J Hoerner ◽  
Lawrence Restaino ◽  
G Anderson ◽  
W Birbari ◽  
...  
Keyword(s):  
Collaborative Study ◽  
Ground Beef ◽  
Culture Method ◽  
The United States ◽  
Test System ◽  
Private Industry ◽  
E Coli ◽  
Inspection Service ◽  
High Level ◽  
The U.S

Abstract Five different food types were analyzed by the Reveal for E. coli O157:H7 8-Hour Test System (Reveal 8) and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method or the U.S. Department of Agriculture Food Safety Inspection Service (FSIS) culture method for the presence of E. coli O157:H7. A total of 27 laboratories representing academia and private industry in the United States and Canada participated. Food types were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. During this study, 1110 samples and controls were analyzed by both the Reveal 8 and by BAM or FSIS by each of the collaborators (2220 samples in total). For each set of samples, 740 were artificially inoculated with E. coli O157:H7, and 370 were uninoculated controls. The Reveal 8 detected 528 presumptive positives of which 487 were confirmed positive by the BAM culture method. In comparison, BAM and FSIS detected 489 of the 740 artificially contaminated samples as positive. In an additional in-house study performed only on chilled and frozen raw ground beef, 240 artificially inoculated samples were analyzed by both the Reveal 8 and by FSIS. The Reveal 8 detected and confirmed 104 samples as positive compared to 79 confirmed positive by FSIS.

Crystal Diagnostics MultiPath System™

2014 ◽  
Vol 97 (6) ◽  
pp. 1592-1600 ◽  
Author(s):  
Curtis H Stumpf ◽  
Weidong Zhao ◽  
Brian Bullard ◽  
Christine Ammons ◽  
Karl I Devlin ◽  
...  
Keyword(s):  
Liquid Crystal ◽  
Laboratory Tests ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Department Of Agriculture ◽  
E Coli ◽  
Independent Laboratory ◽  
Diagnostics System ◽  
Inspection Service ◽  
The U.S

Abstract The Crystal Diagnostics MultiPath System™ provides rapid detection of Escherichia coli O157 in fresh raw ground beef, raw beef trim, and spinach. The Crystal Diagnostics system combines patented Liquid Crystal technology with antibody-coated paramagnetic microspheres to selectively capture and detect E. coli O157 in food matrixes. This is the only liquid crystal-based biosensor commercially available for the detection of pathogens. The Crystal Diagnostics system expeditiously provides the sensitivity and accuracy of the U.S. Department of Agriculture Food Safety Inspection Service (USDA-FSIS) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) methods for detecting as low as one CFU of E. coli O157 per 375 g of raw ground beef and raw beef trim, or 200 g of raw spinach. An internal inclusivity validation demonstrated detection of all 50 tested strains of E. coli O157. The internal and independent laboratory tests demonstrate that the method is rapid and sensitive for detecting of E. coli O157 in fresh raw ground beef, beef trim, and spinach.

scholarly journals In-House Validation Study of the DuPont Qualicon BAX System Q7 Instrument with the BAX System PCR Assay for Salmonella (Modification of AOAC Official MethodSM 2003.09 and AOAC Research Institute Performance-Tested MethodSM 100201)

2009 ◽  
Vol 92 (3) ◽  
pp. 989-994 ◽  
Author(s):  
George Tice ◽  
Bridget Andaloro ◽  
H Kirk White ◽  
Lance Bolton ◽  
Siqun Wang ◽  
...  
Keyword(s):  
Validation Study ◽  
Reference Method ◽  
Pcr Assay ◽  
Paired Samples ◽  
Reference Methods ◽  
Comparable Performance ◽  
Aoac Official ◽  
Inspection Service ◽  
Positive Results ◽  
The U.S

Abstract In 2006, DuPont Qualicon introduced the BAX<sup/> system Q7 instrument for use with its assays. To demonstrate the equivalence of the new and old instruments, a validation study was conducted using the BAX system PCR Assay for Salmonella, AOAC Official MethodSM 2003.09, on three food types. The foods were simultaneously analyzed with the BAX system Q7 instrument and either the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of AgricultureFood Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Comparable performance between the BAX system and the reference methods was observed. Of the 75 paired samples analyzed, 39 samples were positive by both the BAX system and reference methods, and 36 samples were negative by both the BAX system and reference methods, demonstrating 100 correlation. Inclusivity and exclusivity for the BAX system Q7 instrument were also established by testing 50 Salmonella strains and 20 non-Salmonella isolates. All Salmonella strains returned positive results, and all non-Salmonella isolates returned a negative response.

Reveal®E. coli 2.0 Method for Detection of Escherichia coli O157:H7 in Raw Beef

2011 ◽  
Vol 94 (6) ◽  
pp. 1835-1845 ◽  
Author(s):  
Rebecca Hoerner ◽  
Jill Feldpausch ◽  
R Lucas Gray ◽  
Stephanie Curry ◽  
Paul Lewis ◽  
...  
Keyword(s):  
Test Performance ◽  
Test Procedure ◽  
Ground Beef ◽  
Test Sample ◽  
Sample Volume ◽  
Method Performance ◽  
E Coli ◽  
Test Specificity ◽  
Inspection Service ◽  
Positive Results

Abstract Reveal®E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture–Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.

Validation of a Modified ANSR® for E. coli O157:H7 Method for Detection of E. coli O157:H7 in Select Foods

2019 ◽  
Vol 102 (1) ◽  
pp. 96-107
Author(s):  
Susan Alles ◽  
Brooke Roman ◽  
Quynh-Nhi Le ◽  
Edan Hosking ◽  
Wesley Colangelo ◽  
...  
Keyword(s):  
Validation Study ◽  
Laboratory Testing ◽  
Ground Beef ◽  
Nucleic Acid Amplification ◽  
Method Performance ◽  
E Coli ◽  
Independent Laboratory ◽  
Accelerated Stability ◽  
Nucleic Acid Amplification Technology ◽  
The U.S

Abstract Background: The ANSR method is based on isothermal nucleic acid amplification technology. The modifications to assay components improve sensitivity of the assay and robustness of the internal positive control. Objective: A Performance Tested MethodSM validation study was conducted to assess performance of a modified version of the ANSR® for Escherichia coli O157:H7 method. Methods: The validation study included inclusivity/exclusivity, matrix, robustness, accelerated stability, and independent laboratory testing. Results: In inclusivity testing of 55 strains of E. coli O157:H7 and E. coli O157:NM variants, all strains produced positive results. In exclusivity testing of 41 strains including E. coli of other serotypes and bacteria of closely related genera, all strains produced negative results. In matrix testing of beef trim, raw ground beef, spinach, and sprout-irrigation water, ANSR method performance was compared with that of the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook or the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures. Conclusions: all trials, ANSR method performance was not statistically different from that of the reference methods. Results of independent laboratory testing of ground beef corroborated those of internal testing. Introducing modest changes to three assay operating parameters did not materially affect ANSR method performance. Finally, accelerated stability testing results of three independently manufactured lots of ANSR reagents support a shelf-life of 1 year when stored at 2–8°C.

Detection of Listeria spp. Using ACTERO Listeria Enrichment Media

2014 ◽  
Vol 97 (4) ◽  
pp. 1127-1136
Author(s):  
David Claveau ◽  
Sergiy Olishevskyy ◽  
Michael Giuffre ◽  
Gabriela Martinez
Keyword(s):  
Stainless Steel ◽  
Environmental Samples ◽  
Incubation Temperature ◽  
Reference Method ◽  
Selective Medium ◽  
Single Step ◽  
Department Of Agriculture ◽  
Steel Surfaces ◽  
Inspection Service ◽  
The U.S

Abstract ACTERO™ Listeria Enrichment Media (ACTERO Listeria) is a selective medium developed for a single-step recovery and enrichment of Listeria spp. from environmental samples. Robustness testing of the ACTERO Listeria medium demonstrated good performance when minor changes were introduced to the incubation temperature and time. All 54 Listeria strains tested, representing the most frequently isolated Listeria species from food (L. monocytogenes, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi), were successfully enriched in ACTERO Listeria. None of the 30 nontarget strains tested in the exclusivity study was recovered after incubation in ACTERO Listeria. Recovery of Listeria was consistent across three independently produced lots of the ACTERO Listeria, and the prepared medium was stable for 45 days when stored at 4°C in the dark. Matrix studies performed with environmental sponge samples from plastic and stainless steel surfaces demonstrated similar recovery of Listeria spp. in a single-step enrichment using ACTERO Listeria from plastic, and significantly better recovery from stainless steel surfaces when compared to the U.S. Department of Agriculture-Food Safety and Inspection Service reference method. The results of this study prove that ACTERO Listeria Enrichment Media can be effectively used in replacement of the two-step enrichment suggested by the reference method without affecting the recovery of Listeria spp. from environmental samples.

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