AOAC Research Institute Research Articles

Modification of BIOFISH 300 HIS for Determination of Histamine in Fish and Fish Products: AOAC Performance Tested MethodSM 051604.

The BIOFISH 300 HIS method for the quantification of histamine in fish and fish products was validated by the AOAC Research Institute and granted Performance Tested MethodSM certification in 2016. The method is based on the use of an electrochemical reader (BIOFISH 300 device) together with disposable gold electrodes (Biotest) modified with a specific enzyme that oxidizes the histamine molecules and produces a detectable and quantifiable electric current signal. It was validated for raw fish (tuna, mackerel, sardine, and anchovy), cooked fish (tuna), canned fish (tuna in water, tuna in oil, mackerel in tomato sauce, and pickled sardine), and salted fish (canned salted anchovy). The validated ranges were 30-150 mg/kg histamine for canned salted anchovy and 10-200 mg/kg histamine for all other matrices. The objective of the present report is to validate some method modifications, namely the use of a new reader (BIOFISH 3000), the inclusion of new quantification ranges, new matrixes in the certified claims, a histamine reference solution (Verifying Solution) that will enable the user to perform a system verification, and also the inclusion of the kit ASC1-10 for the elimination of ascorbic acid from sample extract. New quantification ranges can be obtained by diluting the sample appropriately into the measurement cuvette, thereby adjusting the concentration to the internal calibration range of the device. New matrixes such as fish meal, preserved salted anchovy, and fish with added ascorbic acid were validated. The new digitalized reader enables new functionalities such as the use of an intuitive app and cloud storage of the results, with no changes in the data acquisition and processing. As the use of the new reader does not imply any change at the electrochemical readout level, all assays were performed using the BIOFISH 3000 device. The method was shown to be linear in all the quantitation ranges configured. Accuracy and recovery studies over a wide range of matrixes showed that the method yields comparable results to the reference method used. Stability and product consistency testing of kit components demonstrated that the system produces accurate results when expiration dates from the manufacturer are met. All modifications made to the BIOFISH 300 HIS method, PTM 051604, have been validated satisfactorily, under criteria of the AOAC Research Institute. The use of the BIOFISH 300/3000 HIS method is ideal as a routine histamine control method for the fish industry. After a simple aqueous extraction of the sample, results are obtained in less than 3 minutes. All data generated by the new BIO3000 biosensor are sent to a web platform, allowing new functionalities to the user. All these technical capabilities represent a competitive advantage over other existing technologies.

Emergency Response Validation of the Soleris® Direct Yeast and Mold Method for Detection of Yeast and Mold in Dried Cannabis Flower: AOAC Performance Tested MethodSM 051301.

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. 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. 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. 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. Results indicate that the Soleris method is an effective procedure for detection of yeast and mold in dried cannabis flower. With the Soleris method, results are available within 72 h compared with the 5-7 days required for microbiological culture methods.

Validation Study for VERIPRO® SARS-CoV-2 Env Assay for the Detection of SARS-CoV-2 from Stainless-Steel Environmental Surface Swabs: Emergency Response Validation—AOAC Performance Tested MethodSM 122001

BackgroundThe VERIPRO SARS-CoV-2 Env assay uses reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2, the causative agent of COVID-19, from stainless steel environmental sample swabs.ObjectiveTo validate the VERIPRO SARS-CoV-2 Env assay as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested MethodSM program.MethodsThe VERIPRO SARS-CoV-2 Env assay was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms). The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020).ResultsResults of the in silico analysis demonstrated the specificity of the method in being able to detect target sequences and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the reference method.ConclusionsThe VERIPRO SARS-CoV-2 Env assay is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on food contact surfaces.HighlightsThe VERIPRO SARS-CoV-2 Env assay can be performed without the need for an optional RNA purification step to detect SARS-CoV-2 from environmental surfaces.

Detection of SARS-CoV-2 Virus on Stainless-Steel Surfaces: AOAC Performance Tested MethodSM 022102.

BackgroundThe SureFast® SARS-CoV-2 PLUS Test is a reverse transcription qPCR (RT-qPCR) assay for the direct, qualitative detection of novel coronavirus (SARS-CoV-2) RNA from stainless-steel environmental sample swabs.ObjectiveTo validate the SureFast SARS-CoV-2 PLUS Kit as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested Method(s)SM program.MethodThe SureFast SARS-CoV-2 PLUS Kit was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms) using the ThermoBLAST program. The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020).ResultsResults of the in silico analysis demonstrated 99.99% selectivity of the method in being able to detect target sequences of the known CoV-2 genomes and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the PCR detection reference method.ConclusionsThe SureFast SARS-CoV-2 PLUS Kit is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on stainless-steel contact surfaces.HighlightsSureFast SARS-CoV-2 PLUS test method is highly specific for primer/probe binding to the E target genome region for the SARS-CoV-2 virus, 99.99% binding specificity using in silico analysis.

Validation of the COVID-19 Indoor Test™ by Phylagen for Detection of SARS-CoV-2 Virus on Stainless-Steel Surfaces: AOAC Performance Tested MethodSM 122004.

BackgroundThe COVID-19 Indoor Test™ by Phylagen uses a real-time PCR Assay to detect nucleic acid from SARS-CoV-2, the causative agent of COVID-19, which is extracted from swabs sampled from environmental surfaces. This information can be used to detect the presence of the virus in indoor environments.ObjectiveTo validate the COVID-19 Indoor TestTM by Phylagen as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested Method(s)SM program.MethodThe COVID-19 Indoor Test by Phylagen assay was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms. The candidate method was also evaluated in an unpaired matrix study design for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (Revision 4, Effective 6/12/2020).ResultsResults of the in silico analysis demonstrated the specificity of the method in being able to detect SARS-CoV-2 target sequences and discriminate them from near-neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the reference method (2 × 103 GU/2 × 2” test surface).ConclusionsThe COVID-19 Indoor Test by Phylagen is a rapid and accurate method that can be utilized to monitor the presence of SARS-CoV-2, the causative agent of COVID-19, on stainless-steel surfaces in built environments.HighlightsThe COVID-19 Indoor Test by Phylagen assay performed significantly better than the reference method when used to detect SARS-CoV-2 from environmental surfaces.

Comparison of the Modified Centers for Disease Control and Prevention 2019-Novel Coronavirus Real-Time RT-PCR Method for Detection of Infectious and Heat-Inactivated Virus on Stainless Steel.

BackgroundInfectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was used in the validation of methods for detection of SARS-CoV-2 on stainless steel surfaces in the AOAC Research Institute Emergency Response Validation project. Handling infectious virus requires Biosafety Level (BSL)-3 facilities.ObjectiveTo compare the recovery and detection of infectious and heat-inactivated (65 °C for 30 min) SARS-CoV-2 from stainless steel by the modified US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Diagnostic Panel.MethodsViral stocks were diluted in viral transport medium and deposited onto stainless steel test areas at 2 x 103 and 2 x 104 genomic copies for low and high, respectively. Test areas were sampled, and aliquots of the resulting test solutions analyzed by RT-qPCR according to the CDC method. Results were analyzed by Probability of Detection (POD) statistics.ResultsThe low level, where fractional positive results (25–75%) are expected, yielded PODI = 0.80 (0.58, 0.92) for the infectious virus and PODHI = 0.15 (0.05, 0.36) for the heat-inactivated virus. The bias, dPODHI = -0.65 (-0.80, -0.35), demonstrated a statistical difference between infectious and heat-inactivated virus detection. No difference was observed at the high inoculation level.ConclusionDespite the statistical difference observed, the use of the heat-inactivated virus is a viable alternative for matrix extension studies using a method comparison study design.HighlightsThe use of heat-inactivated SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies.

Validation Study of BioSystems® Y15 Histamine Dehydrogenase Kit for the Detection of Histamine in Fish and Fishery Products: AOAC Performance Tested MethodSM 072001.

BackgroundBioSystems has developed a histamine kit for automated procedure, through the use of a Y15 analyzer, for quantification of histamine in fish and fishery products.ObjectiveValidate the method under the specific guidelines of the AOAC Research Institute Performance Tested MethodSM (PTM) program.MethodSamples are extracted with boiling water. The enzymatic method is based on histamine dehydrogenase that catalyzes the oxidation of histamine in the presence of an electron mediator that reduces a dye that is measured at 420 nm. The increase of absorbance is proportional to the histamine concentration. Dispensing of reagents and sample, absorbance readings, calibration, and calculation of results are performed automatically in the analyzer BioSystems Y15.ResultsThe linearity ranges from 0 to 200 mg/kg (r2 > 0.99). The LOQ is 10 mg/kg in all the matrixes. Recoveries range from 75 to 107% at concentrations from 5 to 200 mg/kg, with repeatability precision values between 0.8 and 5.5%. Comparisons with the HPLC reference method shows a good correlation. Cross-reactivity of the assay is negligible for all biogenic amines tested except for agmatine (6.3%). Product consistency was verified by validating lot-to-lot variations and variations within the same lot. Shelf life was verified by real-time stability testing during 40 months at 2–8°C. No differences in histamine detection were observed in robustness testing, in which minor changes are introduced to the assay protocol.ConclusionsThe automated, simple, and rapid BioSystems Y15 Histamine Dehydrogenase Kit has been successfully validated.HighlightsThe method is qualified for PTM certification No. 072001.

Reveal Q+ MAX® for Detection of Total Af latoxin in Corn, Almonds, Pistachios, Walnuts, and Peanuts.

Background: The Reveal Q+ MAX for Aflatoxin is a lateral flow immunochromatographic test intended for quantitative analysis within 6 min after aqueous extraction. Objective: Work was conducted to validate the performance of the Reveal Q+ MAX for Aflatoxin method in selected corn and nut matrixes. Methods: This method was validated under the requirements of the AOAC Research Institute Performance Tested MethodSM program. Five matrixes, including corn naturally contaminated with aflatoxin at 0, 5.2, 21.0, 51.6, 103.6, and 282 ppb as well as peanuts, pistachios, walnuts, and almonds spiked at 0, 5, 20, 50, and 300 ppb were analyzed. Results: Average percentage recoveries of the added aflatoxin from the matrixes ranged from 80.8 to 116.9%. Average LOD for all matrixes is 2 ppb and LOQ is 7 ppb. With the exception of sample size for almonds, robustness trials demonstrated that deliberate changes to the assay parameters minimally affected the Reveal Q+ MAX assay performance. Finally, stability results from three independently manufactured lots support Reveal Q+ MAX for Aflatoxin performance consistency and shelf-life of 18 months when stored at room temperature. Conclusions: This study appropriately validates the Performance Tested MethodSM claim for corn and selected nut matrixes on Reveal Q+ MAX for Aflatoxin, an aqueous lateral flow test kit. Highlights: Aqueous lateral flow test kit detects total aflatoxin between 80 to 120% yield with an LOD of 2 ppb.

Method Modification to Extend the Matrix Claim of the Thermo Scientific RapidFinder Salmonella species, Typhimurium, and Enteritidis Multiplex PCR Kit.

Background: The Thermo Scientific RapidFinder™ Salmonella species, Typhimurium and Enteritidis Multiplex PCR Kit is a real-time multiplex PCR assay for the detection and differentiation of Salmonella species, Salmonella Typhimurium, and S. Enteritidis from poultry, pork, and environmental samples. The method has previously been granted certification as Performance Tested Method SM (PTM) 081701, validated according to the AOAC Research Institute (RI) PTM program for poultry (chicken thighs with skin, chicken wings with skin, and chicken nuggets), raw pork sausage matrixes, and stainless steel environmental surface sponges. Objective: This report details the method modification study to validate ground turkey (375 g sample size), chicken carcass rinse, and shell egg matrixes. Methods: The candidate method was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual Chapter 5 for shell eggs and the U.S. Department of Agriculture Food Safety and Inspection Service's Microbiology Laboratory Guidebook 4.09 for ground turkey (375 g) and chicken carcass rinse matrixes. Results: The statistically significant differences found between the candidate and reference methods upon analysis by probability of detection were in favor of the candidate method. Inclusivity and exclusivity testing demonstrated that the RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Kit was able to detect all the major groups of Salmonella. All exclusivity isolates were correctly excluded. Conclusions: The data presented in this report show that the candidate is suitable for the detection and differentiation of Salmonellae from shell egg, chicken carcass rinse, and ground turkey (375 g) matrixes. Highlights: Thermo Scientific RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Kit (candidate method) matrix claims extended to include ground turkey (375 g), chicken carcass rinse and shell egg samples.

Validation Study of Histamine Test for the Determination of Histamine in Selected Fish Products.

Background: Since prescribed limits for histamine in fish have been set by various regulatory bodies around the world, the rapid, specific and easy determination of histamine is in high demand. Objective: The enzymatic histamine assay developed by Kikkoman Biochemifa Co. was validated. Methods: Fresh and frozen raw tuna, canned tuna in oil and water, and anchovy fish sauce were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodSM (PTM) program. Results: Good linearity (R² > 0.9997) for histamine standard solutions, recovery rates (90.3-125.2%), and repeatability precisions (RSDr < 10%) were shown for all spiked tested matrixes. The claimed LOQ values, 20 and 10 mg/kg for solid fish and 160 and 80 mg/kg for fish sauce using a 1 and 2 cm optical path length spectrophotometer, respectively, were validated. The cross-reactivity test showed no positive interference of other biogenic amines, except for agmatine and putrescine. Moreover, no inhibition was confirmed among the 12 biogenic amines. Stability data supported the shelf life (42 months at 4°C), lot-to-lot consistency was demonstrated, and the method was shown to be robust. Independent laboratory testing using canned tuna in oil demonstrated that the recovery ranged from 93.5 to 124.7%, and the RSDr at all spike levels was <20%. Conclusions: The simple and rapid enzymatic histamine assay method has been successfully validated. Highlights: This histamine quantitative assay kit was qualified for PTM certification No. 041802.

Validation of Workflow Changes, Phage Concentration and Reformatted Detection Threshold for the Sample6 DETECT/L™ Test: Level 3 Modification.

The AOAC Research Institute Performance Tested MethodsSM Program certified Sample6 DETECT/L™ in April 2014 (Certification No. 041401) for the detection of Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. marthii, L. welshimeri) on stainless steel environmental surfaces. A modification was approved in January 2016, increasing the concentration of sanitizer-neutralizing reagents in detection reagents, increasing the number of phage in the detection solution, and increasing the sample test volume. Moreover, changes to reduce the number of negative controls and add compatibility with polyurethane sponges were also approved. In this modification, to ensure that DETECT/L continues to meet performance expectations, Sample6 evaluated workflow changes to enhance sensitivity and the ease-of-use of the assay. Changes to the phage concentration and detection threshold, plus the inclusion of a confirmation step (DETECT Check), were validated to obtain better accuracy and optimize assay performance. Inclusivity, exclusivity, and robustness testing were conducted by Sample6 to evaluate the changes. A third-party laboratory compared the DETECT/L assay and the U.S. Department of Agriculture reference method in a stainless steel environmental surface matrix study. The data presented in this report demonstrate that the changes proposed to the DETECT/L assay meet or exceed the performance in the current configuration.

The Validation of the Sample6 DETECTTM HT/L for AOAC Research Institute.

Listeria spp. are an important foodborne human pathogen because of their ability to cause disease and high mortality in individuals, particularly pregnant women, neonates, the elderly, immunocompromised individuals, and children. The Sample6 DETECTTM HT/L Kit is a semi-automated qualitative pathogen detection system designed to detect Listeria spp. (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. marthii) in environmental samples using the Sample6 BioIlluminationTM technology. The study was done to evaluate the Sample6 DETECT HT/L Kit. The assay was evaluated for inclusivity, exclusivity, robustness, product consistency, and stability, and a matrix study of one environmental surface. The performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration reference culture method for Listeria using an unpaired study design. The Sample6 DETECT HT/L assay correctly identified all 50 inclusivity isolates and correctly excluded all 30 nontarget strains evaluated. The assay was not affected by minor variations in incubation temperature and time, or sample volume. Results across three production lots spanning the shelf life of the assay were consistent. In the matrix study, the Sample6 DETECT HT/L for Listeria correctly identified each test portion for the presence or absence of Listeria, and there were no statistically significant differences between candidate and reference method results. The data collected in this study demonstrate that the Sample6 DETECT HT/L assay is a reliable method for the detection of Listeria spp. on stainless-steel environmental surfaces after 22h of enrichment.

Validation Study of MaxSignal® Histamine Enzymatic Assay for the Detection of Histamine in Fish/Seafood.

Bioo Scientific Corp. has developed a rapid enzymatic quantitative assay for the determination of histamine in seafood. Fresh/frozen tuna, canned tuna, pouched tuna, and frozen mahi mahi samples were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodsSM program. Recoveries ranged from 82 to 107% at concentrations ranging from 6 to 72 ppm, with RSDr values between 0.8 and 6.5% (6-72 ppm). The linearity of the assay ranged from 0 to 108 ppm, with R2 values exceeding 0.99. The LOD was 0.9 ppm and the LOQ was 2.6 ppm for frozen tuna, which gave the lowest background level of contaminant. Cross-reactivity of the assay was tested against 14 other biogenic amines and was found to be minimal for all (<0.5%), except for agmatine (4.1%) and putrescine (0.9%). There was no observable interference from any tested biogenic amines. Product consistency was verified by validating lot-to-lot variations and variations within the same lot. Overall recoveries for all tested matrixes were within the acceptable range (80-120%). A 1-year claimed shelf life of the kit at 4°C was verified by accelerated stability study data collected on days 1, 15, and 32 at 25°C and by real-time stability testing at 1-month, 6-month, and 1-year at 4°C. No difference in histamine detection was observed in ruggedness testing, in which minor changes were introduced to the assay protocol. Good agreement was observed between AOAC Official MethodSM 977.13 and the MaxSignal® Histamine Enzymatic Assay method. Independent laboratory testing demonstrated that the MaxSignal method works with the same precision in the hands of minimally trained technicians as with the expert method developers. This study validates the performance of Bioo Scientific's rapid enzymatic method.

Allergic reaction from fingerprint kit attributable to unlabeled gluten, probable wheat flour

Allergic reaction from fingerprint kit attributable to unlabeled gluten, probable wheat flour

Validation of the Applied Biosystems 7500 Fast Instrument for the Detection of Salmonellae with SureTect Salmonella Species PCR Kit.

The Thermo Scientific SureTect™ Salmonella species real-time PCR assay is a rapid alternative method designed for the detection of salmonellae in a wide range of foods, animal feeds, and production-environment samples. The assay has previously been validated according to the AOAC Research Institute Performance Tested Methods(SM) program using Thermo Scientific PikoReal™ PCR cycler and Thermo Scientific SureTect Software Performance Tested Method 051303). This report details the method-modification study performed to validate an updated assay format, utilizing a reduced target probe concentration and an extension of the PCR cycler platform to enable the use of the kit with a Applied Biosystems 7500 Fast PCR cycler and Applied Biosystems RapidFinder™ Express 2.0 software. During this validation study, a matrix study was conducted on a subset of the method's claimed matrixes, comparing the performance of the modified SureTect Salmonella species kit (a reduced target probe concentration with a 7500 Fast platform) to the reference method detailed in ISO 6579:2002. No significant difference by probability of detection statistical analysis was found between SureTect or International Organization for Standardization methods for any of the matrixes analyzed during the study. Inclusivity and exclusivity studies using the modified method demonstrated accurate results for the 117 Salmonella and 36 non-Salmonella strains tested. Multiple production lots of the newly formatted kit were evaluated and found to be consistent with the current assay. Robustness studies confirmed that the change to the kit had no impact on the assay's performance when alterations were made to method parameters having the greatest potential impact on assay performance.

Validation of the Applied Biosystems 7500 Fast Instrument for Detection of Listeria monocytogenes with the SureTect Listeria monocytogenes PCR Assay

In 2013, the Thermo Scientific™ SureTect™ Listeria monocytogenes Real-Time PCR Assay was certified by the AOAC Research Institute (RI) Performance Tested Methods(SM) program as a rapid method for the detection of L. monocytogenes from a wide range of food matrixes and surface samples. This report details the method modification studies undertaken to extend the analysis of this PCR assay to the Applied Biosystems™ 7500 Fast PCR Instrument and Applied Biosystems RapidFinder™ Express 2.0 software allowing the use of the SureTect assay on a 96 well format PCR cycler in addition to the current workflow, which uses the 24 well Thermo Scientific PikoReal™ PCR Instrument and Thermo Scientific SureTect software. Because this study was deemed by AOAC-RI to be a level 2 method modification study, a representative range of food matrixes covering raw ground turkey, 2% fat pasteurized milk, and bagged lettuce as well as stainless steel surface samples were analyzed with the Applied Biosystems 7500 Fast PCR Instrument and RapidFinder Express 2.0 software. All testing was conducted in comparison to the reference method detailed in International Organization for Standardization (ISO) 6579:2002. No significant difference by probability of detection statistical analysis was found between the SureTect Listeria monocytogenes PCR Assay or the ISO reference method methods for any of the matrixes analyzed during the study.

Matrix Extension Study: Validation of Compact Dry YM for Enumeration of Yeast and Mold in Selected Foods.

Nissui Compact Dry YM was originally certified by the AOAC Research Institute Performance Tested Methods SM (PTM) program (PTM No. 100401) for enumeration of yeasts and molds in fruit products after 7 days of incubation. A matrix extension study, organized by Campden BRI (Chipping Campden, United Kingdom), was conducted to extend the method's claim to cooked deli turkey, fresh whole tomatoes, cheese (Wensleydale), sliced white bread, and mayonnaise. In addition, the method was evaluated at 3 and 7 days to validate the 3 day incubation period. Compact Dry YM is a ready-to-use dry media sheet, containing a cold-soluble gelling agent, selective agents, and chromogenic medium, which are rehydrated by adding 1 mL diluted sample. Yeasts and molds appear as blue colonies, while molds can also have a cottony appearance and the color may vary. Users can obtain total yeast and mold count following 3-7 days of incubation at 25 ± 1°C. Method comparison data for cooked deli turkey, fresh whole tomatoes, cheese (Wensleydale), sliced white bread, and mayonnaise were collected in a single-laboratory evaluation by Campden BRI. A multilaboratory study was conducted on orange juice with 10 laboratories participating including the organizing laboratory. Compact Dry YM was compared to ISO 21527-1:2008, Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of yeasts and molds - Part 1: Colony count technique in products with water activity greater than 0.95, the current standard at the time of this study. Each matrix was evaluated for total yeasts and molds at each contamination level (including an uncontaminated level). In the single-laboratory evaluation (cooked deli turkey, fresh whole tomatoes, cheese, sliced white bread, and mayonnaise), colony counts were logarithmically transformed, and then the data were analyzed at each level for repeatability (sr), RSD of repeatability (RSDr), and mean difference between methods with a 95% confidence interval (CI). A CI outside a range of (-0.5 to 0.5) on the log10 mean difference between methods was used as the criterion to establish a significant statistical difference. In the multilaboratory study on orange juice, after logarithmic transformation the data were analyzed for sr, RSDr, and mean difference with 95% CIs and also for reproducibility (sR) and RSD of reproducibility (RSDR). Regression analysis was performed on all matrixes and reported as r2. Results from Compact Dry YM at 3 and 7 days were compared separately to ISO 21527-1 and then to each other. In the single-laboratory evaluation, five contamination levels were tested. With the exception of cheese, there were few, if any, colonies recovered at the two lowest contamination levels. As a consequence, differences of a few colonies resulted in significant statistical differences indicated between Compact Dry YM (at both 3 and 7 days) and ISO 21527-1 for most of the foods at these levels. At the three higher contamination levels, statistical differences were seen between Compact Dry YM at 3 days and the ISO method for cooked deli turkey at the middle level, tomatoes at the two middle levels, sliced white bread at the two highest levels, and mayonnaise at the middle level. After 7 days, statistical differences remained between Compact Dry and ISO methods, based on the CIs, but mean differences between methods were <0.5 log10 CFU/g for all foods at the three highest contamination levels. When comparing results between Compact Dry YM at 3 and 7 days, statistical differences were indicated for tomatoes and sliced white bread, both in the second highest contamination level. Otherwise, differences between results at 3 and 7 days were small. The sr and RSDr values were similar for Compact Dry YM (both days) and ISO 21527-1, and r2 values were ≥0.84 for all comparisons. In the multilaboratory study for orange juice, there were no statistical differences between methods across 11 data sets. Four contamination levels were tested, and at all levels mean differences between methods were <0.1 log10 CFU/mL, and CIs were smaller than (-0.05, 0.15), well within the acceptance criterion of (-0.5, 0.5). The sr, RSDr, sR, and RSDr were similar for each method, and r2 was 1.0 in all comparisons. Results indicate that Compact Dry YM offers comparable results at 3 days to the ISO standard plating method at 5 days, in a space-saving, easy-to-use format.

Validation of the Applied Biosystems 7500 Fast Instrument for Detection of Listeria Species with the SureTect Listeria Species PCR Assay.

The Thermo Scientific™ SureTect™ Listeria species Real-Time PCR Assay was certified during 2013 by the AOAC Research Institute (RI) Performance Tested Methods(SM) program as a rapid method for the detection of Listeria species from a wide range of food matrixes and surface samples. A method modification study was conducted in 2015 to extend the matrix claims of the product to a wider range of food matrixes. This report details the method modification study undertaken to extend the use of this PCR kit to the Applied Biosystems™ 7500 Fast PCR Instrument and Applied Biosystems RapidFinder™ Express 2.0 software allowing use of the assay on a 96-well format PCR cycler in addition to the current workflow, using the 24-well Thermo Scientific PikoReal™ PCR Instrument and Thermo Scientific SureTect software. The method modification study presented in this report was assessed by the AOAC-RI as being a level 2 method modification study, necessitating a method developer study on a representative range of food matrixes covering raw ground turkey, 2% fat pasteurized milk, and bagged lettuce as well as stainless steel surface samples. All testing was conducted in comparison to the reference method detailed in International Organization for Standardization (ISO) 6579:2002. No significant difference by probability of detection statistical analysis was found between the SureTect Listeria species PCR Assay or the ISO reference method methods for any of the three food matrixes and the surface samples analyzed during the study.

Method Modification Study for the Thermo Scientific SureTect™ Listeria Species Assay-Matrix Extension.

The Thermo Scientific™ SureTect™ Listeria species assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. The assay was originally certified as Performance Tested Methods(SM) (PTM) 071304 in 2013. This report details the method modification study undertaken to extend the performance claims of the assay for matrixes of raw ground turkey, raw ground pork, bagged lettuce, raw pork sausages, pasteurized 2% fat milk, raw cod, pasteurized brie cheese, and ice cream. The method modification study was conducted using the AOAC Research Institute (RI) PTM program to validate the SureTect PCR assay in comparison to the reference method detailed in ISO 11290-1:1996 including amendment 1:2004. All matrixes were tested by Thermo Fisher Scientific (Basingstoke, United Kingdom). In addition, three matrixes (raw cod, bagged lettuce, and pasteurized brie cheese) were analyzed independently as part of the AOAC RI-controlled independent laboratory study by the University of Guelph, Canada. Using probability of detection statistical analysis, there was no significant difference in the performance between the SureTect assay and the International Organization for Standardization reference method for any of the matrixes analyzed in this study.

Matrix Extension Study: Validation of the Compact Dry CF Method for Enumeration of Total Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" CF method, Performance Tested Method(SM) 110401, was originally certified for enumeration of coliform bacteria by the AOAC Research Institute Performance Tested Methods(SM) program for raw meat products. Compact Dry CF is a ready-to-use dry media sheet, containing a cold-soluble gelling agent, a chromogenic medium, and selective agents, which are rehydrated by adding 1 mL of diluted sample. Coliform bacteria produce blue/blue-green colonies on the Compact Dry CF, allowing a coliform colony count to be determined in the sample after 24 ± 2 h incubation. A validation study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom, to extend the method's claim to include cooked chicken, fresh bagged prewashed shredded iceberg lettuce, frozen fish, milk powder, and pasteurized 2% milk. Campden BRI collected single-laboratory data for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Thirteen laboratories participated in the interlaboratory study. The Compact Dry CF method was compared to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current version at the time this study was conducted. Each matrix was evaluated at either four or five contamination levels of coliform bacteria (including an uncontaminated level). After logarithmic transformation of counts at each level, the data for pasteurized whole milk were analyzed for sr, sR, RSDr, and RSDR. Regression analysis was also performed and r(2) was reported. Mean difference between methods with 95% confidence interval (CI) was calculated. A log10 range of -0.5 to 0.5 for the CI was used as the acceptance criterion to establish significant statistical difference between methods. In the single-laboratory evaluation (for cooked chicken, lettuce, frozen fish, and milk powder), sr and RSDr values were analyzed and r(2) was reported. Statistical differences were indicated between the Compact Dry CF and ISO 4832 methods in two of five contamination levels tested for lettuce, and in the low contamination levels for cooked chicken, frozen fish, and dry milk powder. For the low levels of cooked chicken, frozen fish, and milk powder, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. In most cases, mean differences between the Compact Dry CF and ISO 4832 methods were small (<0.5 log10), with CIs within the acceptance criterion. The sr and RSDr values were similar for both methods, and r(2) values were >0.94 for all matrixes. In the multilaboratory study, no statistical differences were indicated between methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller for the Compact Dry CF. The r(2) value was 0.99. The Compact Dry CF method offers comparable results to ISO 4832 in a space saving, easy-to-use format.