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Objectives: Tumor budding was suggested as a valuable prognostic factor in oral squamous cell carcinoma (OSCC) but lacks a standardized scoring system. This study evaluates tumor budding in OSCC using the scoring system recommended by the International Tumor Budding Consensus Conference (ITBCC) 2016., Materials and Methods: The study included 114 patients with resected OSCC. Tumor budding was evaluated according to ITBCC criteria and assigned to three categories (low, intermediate, and high tumor budding). The associations between tumor budding and clinicopathological parameters were examined and survival rate analyses were performed by the Kaplan-Meier method. The prognostic value of tumor budding was assessed by Cox regression analysis., Results: Significant correlations of tumor budding with clinicopathological parameters including lymph node metastasis, grade, stage, perineural and lymphovascular invasion, and local recurrence were found. Intermediate and high tumor budding were significantly and independently associated with worse disease-free survival. High tumor budding was identified as an independent prognostic factor for disease-specific and overall survival., Conclusions: The ITBCC scoring system represents a simple, feasible, and reproducible method to evaluate tumor budding in OSCC. Tumor budding, according to ITBCC criteria, showed its prognostic value in resected OSCC, and its incorporation into the histopathological reporting guidelines should be considered., (© 2024 John Wiley & Sons Ltd.)

Background: The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method, Performance Tested Method  SM (PTM) 122302, is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica  1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately., Objective: The objective of the study was to validate the MDA2-SE/ST method for detection of an additional monophasic variant of ST, S. enterica  1,4,[5],12:-:1,2., Methods: A single strain of S. enterica  1,4,[5],12:-:1,2 was tested as part of a seven-strain inclusivity/exclusivity test panel. Strains were tested after growth in two enrichment media used in the method, buffered peptone water (BPW) and buffered peptone water-ISO formulation (BPW-ISO), and at two incubation temperatures, 35 ± 2 °C and 41.5 ± 1 °C., Results: S. enterica  1,4,[5],12:-:1,2 produced positive results in the ST assay and negative results in the SE assay in both enrichment media and at both incubation temperatures. Results for the other six test strains were as expected under all conditions., Conclusions: The MDA2-SE/ST method can detect the monophasic variant S. enterica  1,4,[5],12:-:1,2 as well as S. enterica ser. Typhimurium and the monophasic variant S. enterica  1,4,[5],12:i:-. The MDA2-SE/ST method maintains inclusiveness for S. enterica ser. Typhimurium despite flagellar antigen variation., Highlights: The data were reviewed by the AOAC PTM Program and approval was granted for modification of the MDA2-SE/ST method, PTM 122302., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a rapid, nucleic acid amplification-based test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica  1,4 , [5] , 12: i:-, in select poultry samples. Results for SE and ST are generated separately., Objective: The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in chicken carcass rinse and raw ground chicken (325 g) for Performance Tested Methods  SM (PTM) certification., Methods: The study consisted of inclusivity/exclusivity testing and independent laboratory testing of chicken carcass rinse and raw ground chicken using inoculated matrixes. Data were analyzed using a paired probability of detection model to determine if differences in the number of positive results obtained with the MDA2-SE/ST and reference methods were significant., Results: In inclusivity testing, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1  Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, results for the MDA2-SE/ST and reference methods were in complete agreement for both matrixes., Conclusions: Results of the validation study showed that the MDA2-SE/ST method is an accurate, specific method for detection of SE and ST in select poultry matrixes., Highlights: The data were reviewed by the AOAC PTM Program and approval was granted for certification of the Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium method as PTM 122302., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica  1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately. The method was previously validated for testing of chicken carcass rinse and raw ground chicken (325 g)., Objective: The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in cooked breaded chicken and raw ground chicken (25 g) as a Level 2 modification for Performance Tested Methods  SM certification., Methods: Inclusivity/exclusivity testing and independent laboratory testing of cooked breaded chicken and raw ground chicken (25 g) were conducted., Results: In inclusivity testing with BPW-ISO broth at 41.5 °C, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1  Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, there were no significant differences in the number of positive results obtained with the MDA2-SE/ST and reference methods by probability of detection analysis at the 95% confidence level., Conclusions: Results of the matrix extension study showed that the MDA2-SE/ST method is an accurate method for detection of SE and ST in cooked breaded chicken and raw ground chicken (25 g)., Highlights: Validation of the MDA2-SE/ST method for two additional matrixes provides users with additional capability for specific detection of SE and ST in poultry products., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: The objective of this study was to compare bone invasion type with histopathological, clinical and immunohistochemical prognostic factors., Methods: The study included 49 patients who were treated for oral squamous cell carcinoma. Of which, 30 patients, with presence of bone invasion on histopathology, were divided according to the type of bone invasion (erosive, infiltrative, mixed). Each invasion type was compared to microvascular density using the CD34 marker., Results: The bone invasion was observed in 30 out of 49 patients (61.22%). On McNemar's test, statistically significant association was observed between bone invasion types and histopathological grade. In contrast, no significant correlation was observed between bone invasion type, and tumour volume or nodal metastases. In tumours with bone invasion of the infiltrative type, higher frequency of locoregional relapses was observed. The 5-year survival, since diagnosis, was approximately 60% in the erosive group, 40% in the mixed group, and merely 15% in the infiltrative group., Conclusion: Peritumoural microvascular density was not significantly related to bone invasion types. Whereas, a significantly higher intratumoural microvascular density was observed in infiltrative type of the bone invasion, when compared to the erosive and mixed type., Competing Interests: The authors report no conflicts of interest in this work.

Background: Reveal® 3-D for Gluten is an immunochromatographic assay for the qualitative detection of gluten in environmental samples. The test uses monoclonal antibodies reactive to prolamins in wheat., Objective: The objective of the study was to validate the Reveal 3-D test for detection of gluten in clean-in-place rinse and swabs from a stainless steel surface., Methods: Elements of the study included food selectivity and interference testing, matrix testing, an assay robustness study, and reagent stability/lot-to-lot consistency testing. Wheat flour was used as the spiking material for all matrixes., Results: In selectivity and interference testing, nine target matrixes all tested positive and 36 of 39 non-target matrixes tested negative. Almond flour, sesame flour, and cornstarch produced positive results as 100% commodities; reactivity can be eliminated with dilution or by testing without use of food extraction buffer, which is not a standard part of the environmental testing method. With a gluten spike at 9.3 mg/kg, chestnut flour, guar gum, and xanthan gum as 100% commodities inhibited the ability of the assay to detect gluten when tested without dilution. In quaternary ammonium clean-in-place rinse and swabs from stainless steel, 100% positive results were obtained at levels of 2.8 mg/kg and 4.7 µg/100 cm2, respectively. Results of independent laboratory testing of swabs from stainless steel supported those of internal trials. Robustness testing showed that introducing variations to three operating parameters simultaneously had no adverse effect on assay performance. In the reagent stability study, data supported kit expiration dating of 11 months., Conclusion: Results of the current study show that the Reveal test is an accurate and reliable method for qualitative detection of gluten in select clean-in-place rinse and environmental samples., Highlights: The Reveal test was able to detect gluten at levels of 2.8 ppm in clean-in-place rinse and 4.7 µg/100 cm2 in swabs from stainless steel., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The Soleris®Enterobacteriaceae vial is a growth-based, automated method for detection of bacteria of the family Enterobacteriaceae in foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner, in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. The Soleris method was granted AOAC Performance Tested MethodSM (PTM) status for select foods after successful completion of a validation study (PTM 121901)., Objective: The objective of this study was to validate the method for the detection of Enterobacteriaceae in dried cannabis flower [>0.3% delta-9-tetrahydrocannabinol (THC)]., Methods: The matrix study included comparison of Soleris method presumptive results to confirmation from the Soleris vials, and comparison of the Soleris confirmed results to those of the ISO 21528-2:2017 colony count method. Test materials at four different levels of contamination ranging from 7.8 to 3500 CFU/g were tested at three dilutions, corresponding to test thresholds., Results: Probability of detection analysis at P < 0.05 showed there were no significant differences between Soleris presumptive and confirmed results, and no significant differences between Soleris confirmed and ISO 21528-2:2017 results., Conclusion: The results provided evidence that the Soleris Enterobacteriaceae test is an accurate method for detection of Enterobacteriaceae in dried cannabis flower., Highlights: The Soleris Enterobacteriaceae method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 20-24 h., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Soleris® Non-Fermenting Total Viable Count (NF-TVC) is a growth-based, automated method for semiquantitative detection of aerobic, mesophilic microorganisms in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM status for select foods after successful completion of a validation study., Objective: The objective of the current study was to validate the Soleris NF-TVC method for use with dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)]., Methods: The validation consisted of a comparative matrix study in which naturally contaminated dried cannabis flower was tested with the Soleris NF-TVC method and with the AOAC Official Methods of AnalysisSM  966.23 dilution plating method. Multiple lots of dried cannabis flower were obtained, pre-screened for total aerobic, mesophilic viable count levels, and blended to produce test materials at four different levels of contamination ranging from 1.0 × 103 to 2.2 × 105 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant., Results: For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in performance comparing the Soleris and reference dilution plating methods as determined by probability of detection analysis at P < 0.05., Conclusions: It is concluded that the Soleris NF-TVC method is an accurate and effective method for detection of aerobic, mesophilic microorganisms in dried cannabis flower., Highlights: The Soleris NF-TVC method provides cannabis industry quality control personnel with an effective method for analysis of dried cannabis flower and produces results in 24-48 h., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The Soleris® Coliform Vial is a growth-based, automated method for detection of coliform bacteria in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM certification for select foods after successful completion of a validation study., Objective: The objective of the current study was to validate the Soleris coliform method for use with dried cannabis flower (>0.3% delta 9-tetrahydrocannabinol)., Methods: A comparative matrix study was performed in which naturally contaminated dried cannabis flower was tested with the Soleris coliform method and with the U.S. Food and Drug Administration Bacteriological Analytical Manual solid medium method. Multiple lots of dried cannabis flower were obtained, pre-screened for coliforms, and blended to produce test materials at four different contamination levels ranging from 4.5 to 1600 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant., Results: For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in the number of positive results obtained with the Soleris and cultural plating methods as determined by probability of detection analysis at P < 0.05., Conclusion: The Soleris coliform test is an accurate method for detection of coliform bacteria in dried cannabis flower., Highlights: The Soleris coliform method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 18-24 h., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Soleris® Direct Yeast and Mold (DYM) is a growth-based, automated method for detection of yeast and mold in select foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested., Objective: The objective of this study was to validate the method for testing of dried cannabis flower. The validation was conducted under the Emergency Response Validation program of the AOAC Research Institute., Method: The study included inclusivity and exclusivity testing, in particular testing of yeast and mold species associated with cannabis, and a matrix study in which Soleris method presumptive results were compared with Soleris confirmed results using Dichloran Rose-Bengal Chloramphenicol (DRBC) agar for confirmation. Samples at four different levels of natural yeast and mold contamination were tested at two test thresholds., Results: In inclusivity testing, all 63 yeast and mold strains tested produced positive results within the specified test duration of 72 h. In exclusivity testing, 36 of 37 strains tested produced no detection within 72 h. In matrix testing, there were no significant differences between Soleris presumptive and confirmed results for any contamination level or test threshold as determined by probability of detection analysis., Conclusions: Results indicate that the Soleris method is an effective procedure for detection of yeast and mold in dried cannabis flower., Highlights: With the Soleris method, results are available within 72 h compared with the 5-7 days required for microbiological culture methods., (© The Author(s) 2021. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Soleris®E. coli is an automated, growth-based method for detection and semi-quantitative determination of Escherichia coli in foods. The method can be used in dilute-to-specification (threshold) or presence/absence modes., Objective: The objective of the study was to validate four modifications to the method: (1) a change in the vial detection window plug composition from agar to agarose to improve plug consistency and robustness, (2) a change in pre-enrichment incubation time for presence/absence testing from 6 h to 18-24 h, (3) a change in vial incubation temperature from 44.5 to 43.5°C, and (4) incorporation of a simple direct-from-vial confirmation test as an alternative to traditional procedures., Methods: Elements of the study included inclusivity/exclusivity testing, matrix testing in comparison to the ISO 7251:2005 reference method, reagent stability/lot-to-lot consistency testing, and an independent laboratory study., Results: In inclusivity testing, all 55 Escherichia coli strains tested produced positive results. In exclusivity testing, 30 of 31 strains of other bacterial species produced negative results, the sole exception being a strain of Enterobacter cloacae. In internal and independent laboratory matrix testing of mozzarella cheese, condensed milk, pasteurized liquid egg, and frozen green beans, results showed no significant differences in performance of the Soleris and reference methods with two exceptions, one in which the Soleris method produced more positive results, and one in which the reference method produced more positive results., Conclusion: Performance characteristics of the modified Soleris E. coli method are consistent with those of the original validated method., Highlights: The Soleris E. coli method offers improvements in ease of use and reagent robustness., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: One Broth One Plate for Salmonella (OBOP Salmonella) is a rapid and simple method for detection of Salmonella spp. in food and environmental samples using traditional culture methodology. The method utilizes single-step enrichment followed by plating to a selective/differential, chromogenic agar., Objective: The purpose of the validation study was to measure the effectiveness of the OBOP Salmonella method in comparison to reference culture procedures., Method: Performance of the OBOP Salmonella method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference method for queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, and sponge samples from a stainless steel surface, or to that of the U.S. Department of Agriculture Microbiology Laboratory Guidebook Chapter 4.10 method for raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. Inclusivity/exclusivity, robustness, and stability/lot-to-lot consistency testing was also performed., Results: In the matrix study, there were no statistically significant differences in performance between the OBOP Salmonella and reference methods, as determined by probability of detection analysis (P < 0.05), for any of the matrixes examined. All 104 Salmonella spp. strains produced positive results in inclusivity testing, and all 33 non-salmonellae exclusivity strains tested negative with the OBOP Salmonella method., Conclusions: Results of the validation study show that the OBOP Salmonella method is a reliable procedure for detection of Salmonella spp. in select matrixes. The method is simple to perform, requires no specialized equipment, and produces results in as little as 37 h., Highlights: The OBOP Salmonella method was awarded AOAC PTMSM (#102002) for detection of Salmonella in queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, sponge samples on a stainless steel surface, raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. The method is also approved by MicroVal® for a broad range of foods under certification number 2019LR88., (© AOAC INTERNATIONAL 2021.)

Background: Soleris®Enterobacteriaceae is a growth-based, automated method for detection of Enterobacteriaceae in food., Objective: A study was conducted to validate the Soleris method for detection of Enterobacteriaceae in select foods (pasteurized milk, yogurt, mozzarella cheese, ice cream, dried milk, pasteurized liquid egg, frozen cooked chicken, deli ham, lettuce, and dry dog food) at a threshold of ≥ 10 CFU/g of product., Methods: Inclusivity and exclusivity of the Soleris method were assessed by testing 55 and 38 target and non-target bacterial strains, respectively. Matrix testing was performed with one naturally contaminated and nine inoculated foods. Efficacy of the Soleris method was compared to that of the ISO 21528-2:2017 direct plating reference method using probability of detection analysis. Independent laboratory testing was conducted to verify method performance in two matrixes (yogurt and deli ham). Method robustness, stability, and lot-to-lot consistency of the Soleris reagents were also assessed., Results: Inclusivity of the Soleris test was 91% and exclusivity was 100%. In matrix testing, there were no significant differences in the number of positive results obtained with the Soleris and reference methods for any of the matrixes examined. Overall, of 370 test portions, there were 176 positive results by the Soleris method and 177 positive results by the reference procedure., Conclusions: Soleris Enterobacteriaceae is an effective method for detection of Enterobacteriaceae in the foods evaluated, with performance equivalent to that of the ISO 21528-2:2017 reference method., Highlights: The Soleris method offers the advantages of labor savings and results within 18 h., (© AOAC INTERNATIONAL 2020.)

Background: NeoSeekTM STEC is a single-source, service-based method for detection and identification of select Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7 and STEC of somatic groups O26, O45, O103, O111, O121, and O145. The method is a multiplex molecular method utilizing more than 80 genetic targets to identify STEC in complex matrices such as food enrichment cultures., Objective: A study was conducted to validate the NeoSeek method for detection of select STEC in raw beef trim., Methods: Performance of the NeoSeek STEC method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service reference methods for E. coli O157:H7 and non-O157 STEC for detection of E. coli O157:H7 and E. coli O26:H11 in raw beef trim. Additionally, inclusivity/exclusivity testing and method robustness testing were performed., Results: Results of raw beef trim testing showed no statistically significant differences in performance between the NeoSeek and reference methods in the ability to detect either E. coli O157:H7 or E.coli O26:H11, as determined by probability of detection analysis. Results of inclusivity and exclusivity testing showed 100% expected results with target and nontarget bacteria, with the exception of a single strain of E. coli O157:H7, which was subsequently verified to be stx-negative by PCR. Conclusions and Highlights: NeoSeek STEC is an accurate, reliable method for rapid detection and identification of select STEC in complex populations such as beef trim enrichment cultures., (Copyright ©2019 by AOAC International, Inc. All rights reserved.)

A chemiluminescence assay using a handheld luminometer to measure the activity of alkaline phosphatase was developed that can detect 0.002% or more of unpasteurized milk in various milk products. Evaluation of the assay followed an National Conference on Interstate Milk Shipments (NCIMS)-approved protocol in which aliquots of pasteurized milk products were spiked with raw milk at various levels. Milk products evaluated included skim white milk, 1 and 2% fat content white milk, whole white milk, strawberry-flavored 1% fat content milk, chocolate-flavored 1% fat content milk, half-and-half, and heavy cream. Split samples were prepared, and alkaline phosphatase activities were determined in triplicate on 4 days by three NCIMS-accredited laboratories by the chemiluminescent method and NCIMS-approved reference methods. Equivalence of the chemiluminescent method to the approved reference methods was demonstrated for all eight products evaluated over a range of raw milk concentration from 0 to 0.5%, using criteria established by NCIMS, in which mean results obtained by the three laboratories by the chemiluminescent method were within 1 standard deviation of the mean results obtained by the NCIMS-approved reference methods at each alkaline phosphatase concentration. Procedures for measurement of microbial and reactivated alkaline phosphatase were also established for the method.

Background: Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces., Objective: A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces., Methods: Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model., Results: There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis., Conclusions: The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces., Highlights: Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents., (© Journal of AOAC International.)

Background: Listeria Right Now TM is a novel, enrichment-free molecular method for detection of Listeria spp. in swab samples from environmental surfaces. The test provides results in real time, indicating the current or recent presence of Listeria spp. in the environment. After sampling, the entire contents of the swab are subject to sample processing, releasing large quantities of target ribosomal RNA molecules into the lysate. A portion of the lysate is then tested using the ANSR ® for Listeria isothermal nucleic acid amplification assay. Objective: A Performance Tested Method SM study was conducted to validate the method for detection of Listeria spp. in swab samples from stainless steel and sealed concrete surfaces. Methods and Results: In inclusivity testing, 60 of 60 Listeria spp. strains tested positive. In exclusivity testing, 31 of 31 nontarget bacterial strains tested negative. In LOD testing, the test was able to detect as few as 2 CFU of L. monocytogenes applied to a stainless steel surface. In matrix testing of inoculated stainless steel and sealed concrete surfaces, there were no statistically significant differences in method performance comparing the Listeria Right Now and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures as determined by probability of detection analysis. In robustness testing, modest changes to three assay operating parameters simultaneously did not significantly affect performance of the test. Conclusions and Highlights: Results can be obtained in less than 1 h using the Listeria Right Now test, allowing food industry personnel to take immediate corrective action in the case of Listeria contamination incidents.

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 Method SM 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.

A study was conducted to validate a minor reagent formulation change to the ANSR for Listeria method, Performance Tested MethodSM 101202. This change involves increasing the master mix volume prelyophilization by 40% and addition of salmon sperm DNA (nontarget DNA) to the master mix. These changes improve the robustness of the internal positive control response and reduce the possibility of obtaining invalid results due to weak-positive control curves. When three foods (hot dogs, Mexican-style cheese, and cantaloupe) and sponge samples taken from a stainless steel surface were tested, no significant differences in performance between the ANSR and U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedures were observed for any of the matrixes as determined by probability of detection analysis. Inclusivity and exclusivity testing yielded 100% expected results for target and nontarget bacteria. Accelerated stability testing was carried out over a 7 week period and showed no decrease in assay performance over time.

The AccuPoint Advanced ATP Hygeine Monitoring System was validated by an AOAC International Performance Tested MethodSM on the detection of ATP from stainless steel surfaces. Neogen Corp.'s system is a lightweight, hand-held diagnostic tool used to validate and verify a hygiene program's effectiveness by detecting organic residues remaining on surfaces and in liquids after cleaning. The system is composed of three primary components: an electronic luminometer, fully self-contained single-use samplers, and software. The system is designed to detect adenosine triphosphate (ATP) at set thresholds and to report the measurement in relative light units (RLU). These thresholds are established by a facility to reflect effective cleaning practices. The instrument compares the measured level of ATP with the established threshold and reports the results as pass, marginal, or fail. A linear dose-response in RLU was observed with pure analyte. In the matrix and microbial studies, detection levels varied depending on the matrix and microorganism tested. Independent laboratory trials confirmed pure analyte and matrix observations. Specificity testing of similar, yet different, compounds resulted in 0 RLU for all except 2'-deoxyadenosine 5'-triphosphate sodium salt, which showed markedly reduced reactivity when compared with ATP. Also, interference by these compounds was negligible. When disinfectant residues were evaluated for their effect on the test, cleaners increased RLU output to varying degrees. Stability testing showed consistent results between three independently manufactured lots and stable results through the 9 month shelf-life. Additionally, when three readers were compared using electronic light-emitting diodes as the light source, instrument variability was low (<3%). Robustness testing results provided evidence that temperature affects test performance more than shaking time, and sampler performance improves as the temperature increases to room temperature. These results provided evidence that the AccuPoint Advanced ATP Hygiene Monitoring System produces consistent and reliable data for the evaluation of sanitation program effectiveness on stainless steel surfaces in food processing and food service facilities.

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.

A study was conducted to assess the performance of the Reveal(®) 2.0 Group D1 Salmonella lateral flow immunoassay for use in detection of Salmonella Enteritidis (SE) in raw shell eggs and poultry-associated matrixes, including chicken carcass rinse and poultry feed. In inclusivity testing, the Reveal 2.0 test detected all 37 strains of SE tested. The test also detected all but one of 18 non-Enteritidis somatic group D1 Salmonella serovars examined. In exclusivity testing, none of 42 strains tested was detected. The exclusivity panel included Salmonella strains of somatic groups other than D1, as well as strains of other genera of Gram-negative bacteria. In matrix testing, performance of the Reveal 2.0 test was compared to that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for chicken carcass rinse and to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual for raw shell eggs and poultry feed. For all matrixes evaluated, there were no significant differences in the ability to detect SE when comparing the Reveal 2.0 method and the appropriate reference culture procedure as determined by probability of detection statistical analysis. The ability of the Reveal 2.0 test to withstand modest perturbations to normal operating parameters was examined in robustness experiments. Results showed that the test can withstand deviations in up to three operating parameters simultaneously without significantly affecting performance. Real-time stability testing of multiple lots of Reveal 2.0 devices established the shelf life of the test device at 16 months postmanufacture.

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.

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.

A study was conducted to validate minor reagent formulation, enrichment, and procedural changes to the ANSR(®) Listeria method, Performance-Tested Method(SM) 101202. In order to improve ease of use and diminish risk of amplicon contamination, the lyophilized reagent components were reformulated for increased solubility, thus eliminating the need to mix by pipetting. In the alternative procedure, an aliquot of the lysate is added to lyophilized ANSR reagents, immediately capped, and briefly mixed by vortexing. When three foods (hot dogs, Mexican-style cheese, and cantaloupe) and sponge samples taken from a stainless steel surface were tested, significant differences in performance between the ANSR and U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedures were seen with hot dogs and Mexican-style cheese after 16 h enrichment, with the reference methods producing more positive results. After 24 h enrichment, however, there were no significant differences in method performance for any of the four matrixes tested. Robustness testing was also conducted, with variations to lysis buffer volume, lysis time, and sample volume having no demonstrable effect on assay results. Accelerated stability testing was carried out over a 10-week period and showed no diminishment in assay performance. A second phase of the study examined performance of the ANSR assay following enrichment in a new medium, LESS Plus broth, designed for use with all food and environmental sample types. With the alternative LESS Plus broth, there were no significant differences in performance between the ANSR method and the reference culture procedures for any of the matrixes tested after either 16 or 24 h enrichment, although 24 h enrichment is recommended for hot dogs due to higher sensitivity. Results of inclusivity and exclusivity testing using LESS Plus broth showed that the ANSR assay is highly specific, with 100% expected results for target and nontarget bacteria.

ANSR® Listeria was previously certified as Performance Tested Method(SM) 101202 for detection of Listeria spp. on selected environmental surfaces. This study proposes a matrix extension to the method for detection of Listeria spp. in selected food matrixes. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in less than 50 min, requiring only simple instrumentation. Inclusivity testing was performed using a panel of 51 strains of Listeria spp., representing the species L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. All strains tested were detected by the ANSR assay. Exclusivity testing of 30 strains representing non-Listeria Gram-positive bacteria yielded no evidence of cross-reactivity. Performance of the ANSR method for detection of Listeria spp. was compared to that of reference culture procedures for pasteurized liquid egg, pasteurized 2% milk, Mexican-style cheese, ice cream, smoked salmon, lettuce, cantaloupe, and guacamole. Data obtained in these unpaired studies and analyzed using a probability of detection model demonstrated that there were no statistically significant differences in results between the ANSR and reference culture methods, except for milk at 16 h and cantaloupe. In milk and smoked salmon, ANSR sensitivity was low at 16 h and therefore the recommended incubation time is 24 h. In cantaloupe, ANSR was found to be more sensitive than the reference culture method at both 16 and 24 h in independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in selected food types.

Here we describe results of a study to validate minor reagent formulation changes to the Soleris Direct Yeast and Mold (DYM) automated growth-based method for semi-quantitative detection of yeast and mold in food products. In order to reduce the maximum concentration of the selective agent chloramphenicol in the Soleris reagents, chloramphenicol was removed from the selective supplement and added to the vial growth medium itself. Therefore, both the vial medium and supplement have been reformulated in an alternative version of the method. A probability of detection (POD) statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with plate counts determined using the U.S. Food and Drug Administration Bacteriological Analytical Manual dilution plating procedure. Three matrixes were tested; yogurt, tomato juice, and cocoa powder. POD analysis showed that the percentage of positive Soleris tests at various test thresholds were within the limits predicted by the reference method plate counts for all matrixes evaluated. Real-time stability data on three manufactured lots showed that the modified Soleris vial and supplement are stable for at a minimum of 10 months when stored at 2-8°C. In sum, results presented here demonstrate that the modifications to the Soleris DYM vial and supplement do not impact method performance. The modified Soleris DYM method can be used as an accurate alternative to conventional dilution plating procedures for semi-quantitative determination of yeast and mold at threshold levels, while saving as much as 3 days in analysis time.

NeoFilm Yeast and Mold (Y&M), also known as Sanita-kun Yeasts and Molds, is a simple, effective device used for the enumeration of yeasts and molds. It consists of a nonwoven fabric on which a layer of microbial nutrients is deposited in a film. A 1 mL sample homogenate is applied to the membrane and this, in turn, is incubated for 48-72 h at 25°C. Sample homogenates were prepared using two different diluents for customer convenience: phosphate buffered saline (PBS) and 0.1% peptone water. In comparative testing of breaded chicken nuggets, dry pet food, orange juice concentrate, yogurt, and cake mix, there were statistically significant differences in the counts obtained by the NeoFilm Y&M and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture methods only in the following instances: medium level for orange juice with PBS as diluent and low level for pet food with 0.1% peptone water as diluent, where reference method counts were higher than those of NeoFilm; medium level for cake mix with PBS, and low and medium levels for cake mix with 0.1% peptone water, where NeoFilm produced higher counts than the reference method. In addition to the method comparison study with five matrixes, robustness and stability/lot-to-lot testing were also performed. Results of robustness testing showed no significant effect on results even with perturbation to three assay parameters simultaneously. Results of testing of three lots of devices ranging in age from 2 to 26 months post-manufacture showed no significant differences in performance.

This paper describes the results of a study to validate minor reagent formulation and procedural changes to the ANSR® Salmonella method, AOAC Performance Tested Method™ 061203. In order to improve ease of use and diminish risk of amplicon contamination, the lyophilized reagent components were reformulated for increased solubility, thus eliminating the need to mix by pipetting. In the alternative procedure, an aliquot of the lysate is added to lyophilized ANSR reagents, immediately capped, and briefly mixed by vortex. Results of the validation study with ice cream, peanut butter, dry dog food, raw ground turkey, raw ground beef, and sponge samples from a stainless steel surface showed no statistically significant differences in performance between the ANSR method and the U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture-Food Safety and Inspection Services Microbiology Laboratory Guidebook reference culture procedures. Results of inclusivity and exclusivity testing were unchanged from those of the original validation study; exclusivity was 100% and inclusivity was 99.1% with only a single strain of Salmonella Weslaco testing negative. Robustness testing was also conducted, with variations to lysis buffer volume, lysis time, and sample volume having no demonstrable effect on assay results.

A matrix extension study was conducted to validate the ANSR(®) Salmonella method for use with pasteurized egg products. Four diverse egg product types were tested by the ANSR method and the U.S. Department of Agriculture's Microbiology Laboratory Guidebook reference culture procedure. There were no significant differences in the number of positive test portions by the two methods for any of the products examined. Independent laboratory testing of pasteurized liquid egg also found no significant difference in performance between the ANSR and reference culture procedures. There were no false-positive results obtained in either internal or independent laboratory testing. Inclusivity testing using a new enrichment medium specifically designed for use with pasteurized egg products produced 112 positive results out of 113 Salmonella spp. strains tested, with only a single strain of S. Weslaco testing negative by the ANSR assay. Exclusivity testing of 38 non-salmonellae produced all negative ANSR results. It is concluded that ANSR Salmonella is a reliable, sensitive, and specific method for detection of Salmonella spp. in pasteurized egg products.

A study was carried out to determine the efficacy of the Soleris Direct Yeast and Mold (DYM) automated growth-based method for semiquantitative detection of yeast and mold in a variety of food products. A probability of detection (POD) statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with plate counts determined using the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 18, dilution plating procedure. Fourteen naturally contaminated food products were tested, with Soleris testing performed at three or more threshold levels for each food. Using the POD model, the majority of Soleris test results were in statistical agreement with the reference plating procedures. The exceptions included a single threshold level in yogurt, black pepper, dried fruit, and dry pet food, and two levels in nonfat dry milk and saw palmetto powder. In all but one of these instances, the exception being pet food, the statistical disagreement was due to Soleris estimating a higher level of contamination than the reference method. Results of ruggedness testing showed that the Soleris method produced accurate results even when significant variances in a critical operating parameter, incubation temperature, were introduced. Results of the internal and independent laboratory validation studies showed that the Soleris DYM method can be used as an accurate alternative to conventional dilution plating procedures for evaluation of yeast and mold counts at threshold levels, while saving as much as 72 h in analysis time.

A collaborative study was conducted to evaluate performance of the ANSR for Salmonella assay for identification of Salmonella spp. from colony picks taken from selective/differential agar media. The ANSR Salmonella assay is an isothermal nucleic acid amplification test based on the nicking enzyme amplification reaction chemistry. The test can be completed in less than 40 min including sample preparation. A total of 18 laboratories representing industry, government, academic, and commercial testing laboratories participated in the study. Each collaborator tested up to 84 samples, comprised of colony picks of six Salmonella spp. and six non-salmonellae taken from six selective/differential agar media as well as tryptic soy agar. A total of 1441 analyses were performed, 1416 of which gave the correct identification, for overall accuracy of 98.3%. For identification of Salmonella spp., 755 of 756 tests (99.9%) produced the correct result. For identification of non-salmonellae as such, 661 of 685 assays (96.5%) produced the correct result. Of the 18 laboratories, 15 produced data sets with 99-100% accuracy. The majority of false-positive results were clustered in three laboratories; analysis of raw data suggests procedural difficulties in at least two cases, which may explain the atypical data from these collaborators. The ANSR Salmonella assay can be used as a rapid, accurate adjunct or alternative to biochemical testing for identification of presumptive Salmonella spp. isolates.

This study represents a proposal to extend the matrix claims for the ANSR Salmonella test, Performance Tested Method 061203. The test is based on the nicking enzyme amplification reaction (NEAR) isothermal nucleic acid amplification technology. The assay platform features simple instrumentation, minimal labor, and following a single-step 16-24 h enrichment (depending on sample type), an extremely short assay time of 30 min including sample preparation. Detection is real-time using fluorescent molecular beacon probes. ANSR Salmonella was originally validated for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, and oat cereal, and on stainless steel, plastic, sealed concrete, ceramic tile, and rubber surfaces. The matrixes tested in this study include pet food, ice cream, soy flour, raw almonds, peanut butter, spinach, black pepper, raw frozen shrimp, cocoa powder, and pasteurized dried egg. In unpaired comparative testing there were no statistically significant differences in the number of positive results obtained with the ANSR and the reference culture methods. Enrichment for 16 h was effective for all commodities tested except ice cream, black pepper, dried pasteurized egg, and 375 g samples of dry pet food, for which enrichment for 24 h is indicated.

The Soleris Non-fermenting Total Viable Count method was previously validated for a wide variety of food products, including cocoa powder. A matrix extension study was conducted to validate the method for use with cocoa butter and cocoa liquor. Test samples included naturally contaminated cocoa liquor and cocoa butter inoculated with natural microbial flora derived from cocoa liquor. A probability of detection statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with aerobic plate counts determined using the AOAC Official Method 966.23 dilution plating method. Results of the two methods were not statistically different at any dilution level in any of the three trials conducted. The Soleris method offers the advantage of results within 24 h, compared to the 48 h required by standard dilution plating methods.

ANSR Listeria is a new diagnostic assay for detection of Listeria spp. in sponge or swab samples taken from a variety of environmental surfaces. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in 40 min, requiring only simple instrumentation. In inclusivity testing, 48 of 51 Listeria strains tested positive, with only the three strains of L. grayi producing negative results. Further investigation showed that L. grayi is reactive in the ANSR assay, but its ability to grow under the selective enrichment conditions used in the method is variable. In exclusivity testing, 32 species of non-Listeria, Gram-positive bacteria all produced negative ANSR assay results. Performance of the ANSR method was compared to that of the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of Listeria spp. in sponge or swab samples taken from inoculated stainless steel, plastic, ceramic tile, sealed concrete, and rubber surfaces. Data were analyzed using Chi-square and probability of detection models. Only one surface, stainless steel, showed a significant difference in performance between the methods, with the ANSR method producing more positive results. Results of internal trials were supported by findings from independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in environmental samples.

ANSR Salmonella is a new molecular diagnostic assay for detection of Salmonella spp. in foods and environmental samples. The test is based on the nicking enzyme amplification reaction (NEAR) isothermal nucleic acid amplification technology. The assay platform features simple instrumentation, minimal labor, and, following a single-step 10-24 h enrichment (depending on sample type), an extremely short assay time of 30 min, including sample preparation. Detection is real-time using fluorescent molecular beacon probes. Inclusivity testing was performed using a panel of 113 strains of S. enterica and S. bongori, representing 109 serovars and all genetic subgroups. With the single exception of the rare serovar S. Weslaco, all serovars and genetic subgroups were detected. Exclusivity testing of 38 non-salmonellae, mostly Enterobacteriaceae, yielded no evidence of cross-reactivity. In comparative testing of chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, and oat cereal, there were no statistically significant differences in the number of positive results obtained with the ANSR and the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods. In testing of swab or sponge samples from five different environmental surfaces, four trials showed no statistically significant differences in the number of positive results by the ANSR and the U.S. Food and Drug Administration/ Bacteriological Analytical Manual reference methods; in the trial with stainless steel surface, there were significantly more positive results by the ANSR method. Ruggedness experiments showed a high degree of assay robustness when deviations in reagent volumes and incubation times were introduced.

A study was conducted to determine the efficacy of the Soleris Non-fermenting-Total Viable Count (NF-TVC) automated growth-based method for semiquantitative detection of mesophilic, aerobic microorganisms in a variety of food products. A probability of detection (POD) statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with aerobic plate counts determined using reference dilution plating procedures. Nine naturally contaminated food products were tested, with Soleris testing performed at three or four threshold levels for each food. Using the POD model, all Soleris test results were in statistical agreement with the reference plating procedures with the exception of a single threshold level in two trials with black pepper, and a single threshold level in the independent laboratory trial with cheesecake. Results of ruggedness testing showed that the Soleris method produced accurate results even when minor variances in operating parameters, including sample volume and incubation temperature, were introduced. Results of the internal and independent laboratory validation studies showed that the Soleris NF-TVC method can be used as an accurate alternative to conventional dilution plating procedures for evaluation of microbial counts at threshold levels, while saving 24 h or more in analysis time.

Neogen Corp. developed the Veratox R5 Gliadin test kit for the detection of gliadin based on the monoclonal antibody R5 developed by Enrique Mendez (1). The purpose of this study was to validate the method under the requirements of the AOAC Research Institute Performance Tested Method program. There are two AOAC Official Methods for gluten detection in foods, 991.19 and 2012.01 (2), both of which are ELISAs. With the R5 Mendez method listed in the CODEX Alimentarius as a type 1 method for the detection of gluten in foods, the need to have additional rapid test kits validated by the AOAC Research Institute exists.

A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration, Center for Veterinary Medicine (FDA/CVM) was conducted for a receptor and antibody-based, immunochromatographic method (BetaStar Plus) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels, but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments (NCIMS). Results of the part I (internal) and part II (independent laboratory) dose-response studies employing spiked samples were in close agreement. The test was able to detect all six drugs at the approximate 90/95% sensitivity levels when presented as incurred residues in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing of 1031 control milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar Plus assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

A Performance Tested Method validation study was conducted for a new lateral flow immunoassay (Reveal Listeria 2.0) for detection of Listeria spp. in foods and environmental samples. Results of inclusivity testing showed that the test detects all species of Listeria, with the exception of L. grayi. In exclusivity testing conducted under nonselective growth conditions, all non-listeriae tested produced negative Reveal assay results, except for three strains of Lactobacillus spp. However, these lactobacilli are inhibited by the selective Listeria Enrichment Single Step broth enrichment medium used with the Reveal method. Six foods were tested in parallel by the Reveal method and the U.S. Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) reference culture procedure. Considering data from both internal and independent laboratory trials, overall sensitivity of the Reveal method relative to that of the FDA/BAM procedure was 101%. Four foods were tested in parallel by the Reveal method and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedure. Overall sensitivity of the Reveal method relative to that of the USDA-FSIS procedure was 98.2%. There were no statistically significant differences in the number of positives obtained by the Reveal and reference culture procedures in any food trials. In testing of swab or sponge samples from four types of environmental surfaces, sensitivity of Reveal relative to that of the USDA-FSIS reference culture procedure was 127%. For two surface types, differences in the number of positives obtained by the Reveal and reference methods were statistically significant, with more positives by the Reveal method in both cases. Specificity of the Reveal assay was 100%, as there were no unconfirmed positive results obtained in any phase of the testing. Results of ruggedness experiments showed that the Reveal assay is tolerant of modest deviations in test sample volume and device incubation time.

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.

Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A-E in a variety of food and environmental samples. A Performance Tested Method validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A-E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.

Reveal Salmonella Enteritidis (SE) is a lateral flow-based immunodiagnostic assay used for rapid detection of Salmonella enterica serovar Enteritidis from pooled shell eggs and environmental samples. This assay uses highly specific antibodies to accurately detect S. Enteritidis. Studies were conducted to compare the performance of this test against reference procedures for detection of S. Enteritidis from both pooled shell eggs and environmental samples. Pooled shell eggs were inoculated with low levels ofS. Enteritidis and were enriched according to the procedure prescribed by the U.S. Food and Drug Administration. Uninoculated samples were included in each trial. Reveal SE exhibited 100% sensitivity and 100% specificity in comparison to the reference method in all trials. An abbreviated 48 h/(no hold) enrichment procedure was also developed and validated for detection ofS. Enteritidis from pooled shell egg samples. This shortened enrichment procedure can be used in conjunction with the Reveal SE test and offers a significant enrichment time savings of 96 h. Chi-square analysis revealed that there was no significant difference between the abbreviated Reveal method and the reference procedure for detection ofS. Enteritidis from pooled shell egg samples. Out of 245 natural drag swabs screened internally, only three samples tested Reveal SE positive and were confirmed by the reference procedure, resulting in 100% sensitivity and 100% specificity. An external laboratory screened 147 poultry house environmental samples and obtained 35 Reveal SE confirmed positives for Reveal SE sensitivity of 100% and specificity of 90%. Inoculation trials with drag swabs resulted in 96% sensitivity and 100% specificity. Thus, these data demonstrate that Reveal SE is a highly sensitive and specific assay for the detection of S. Enteritidis from both pooled shell eggs and environmental samples.

Neogen Corp. has developed an improved Veratox histamine test kit for the detection of histamine in tuna tissue. The purpose of this study was to validate the method under the requirements of AOAC Research Institute (RI) Performance Tested Methods. Three AOAC Official Methods for histamine (954.04, 957.07, and 977.13) and one ELISA method have been performance tested by the AOAC RI. The most popular is AOAC Official Method 977.13, the fluorometric method, which is considered the reference method, but is complicated and time-consuming. The need for a rapid ELISA test kit to be validated by the AOAC RI exists.