David Crabtree

Evaluation of the Thermo Scientific™ SureTect™ listeria species assay

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. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested MethodsSM program to validate the SureTect Listeria species Assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996 including amendment 1:2004 in a variety of foods plus plastic and stainless steel. The food matrixes validated were smoked salmon, processed cheese, fresh bagged spinach, cantaloupe, cooked prawns, cooked sliced turkey meat, cooked sliced ham, salami, pork frankfurters, and raw ground beef. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, fresh bagged spinach, and stainless steel surface samples) 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, a significant difference in favour of the SureTect assay was demonstrated between the SureTect and reference method for high level spiked samples of pork frankfurters, smoked salmon, cooked prawns, stainless steel, and low-spiked samples of salami. For all other matrixes, no significant difference was seen between the two methods during the study. Inclusivity testing was conducted with 68 different isolates of Listeria species, all of which were detected by the SureTect Listeria species Assay. None of the 33 exclusivity isolates were detected by the SureTect Listeria species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation, which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.

Method Modification of the Thermo Scientific SureTect Listeria monocytogenes Assay for Raw Meat, Dairy, Produce, and Seafood.

The Thermo Scientific™ SureTect™ Listeria monocytogenes assay is a real-time PCR assay for the detection of Listeria monocytogenes in food and environmental samples, which was certified during 2013 by the AOAC Research Institute (RI) as Performance Tested Method(SM) (PTM) 061302 for a representative range of key food matrixes and production surfaces. This report details the method modification study, which was conducted during 2014, using the AOAC-RI PTM program to extend the validated matrix claims of the assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996, including Amendment 1:2004, to gain certification for raw ground turkey, raw ground pork, pasteurized 2% milk, raw pork sausages, raw cod, pasteurized brie cheese, cooked sliced ham, and bagged lettuce. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, brie cheese, bagged lettuce, and raw cod were analyzed independently by the University of Guelph, Canada, during the AOAC-RI controlled independent laboratory study. Using probability of detection (POD) statistical analysis, a significant difference was demonstrated between the candidate and reference methods for the high spiking level with raw ground pork and brie cheese. For all other matrixes and the low spiked levels for raw ground pork and brie cheese, no significant difference by POD was seen between the two methods during the study.

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 Administrations Bacteriological Analytical Manual Chapter 5 for shell eggs and the U.S. Department of Agriculture Food Safety and Inspection Services 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.

Evaluation of the Thermo Scientific RapidFinder™ Salmonella Species, Typhimurium, and Enteritidis Multiplex PCR Kit

The Thermo Scientific RapidFinder™ Salmonella Species, Typhimurium, and Enteritidis Multiplex PCR Kit (candidate method) is a real-time PCR assay for the detection and differentiation of Salmonella spp., and the serovars S. Typhimurium, and S. Enteritidis from poultry, pork, and environmental samples. The method was validated in comparison to the U.S. Department of Agriculture Food Safety and Inspection Service and the U.S. Food and Drug Administration reference methods. Thermo Fisher Scientific (Basingstoke, United Kingdom) tested all matrixes. In addition, two matrixes were analyzed independently by Q Laboratories, Inc. (Cincinnati, OH). Few statistically significant differences were found between the candidate and reference methods when analyzed by probability of detection. When differences were observed, these were in favor of the candidate method. All 200 inclusivity strains and none of the 45 exclusivity strains were detected, which demonstrated that the RapidFinder Salmonella Species, Typhimurium, and Enteritidis Multiplex PCR Kit was able to detect all the major groups of Salmonella, the less common subspecies of S. enterica, and the rarely encountered S. bongori. None of the exclusivity isolates analyzed were detected. Robustness testing demonstrated that the assay gave reliable performance, with specific method deviations outside the recommended parameters. Accelerated stability testing was conducted, validating the assay shelf life.

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.

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.

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 methods 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 assays 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.

Evaluation of the Thermo Scientific™ SureTect™ Salmonella species Assay

The Thermo Scientific™ SureTect™ Salmonella species Assay is a new real-time PCR assay for the detection of Salmonellae in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested MethodsSM program to validate the SureTect Salmonella species Assay in comparison to the reference method detailed in International Organization for Standardization 6579:2002 in a variety of food matrixes, namely, raw ground beef, raw chicken breast, raw ground pork, fresh bagged lettuce, pork frankfurters, nonfat dried milk powder, cooked peeled shrimp, pasteurized liquid whole egg, ready-to-eat meal containing beef, and stainless steel surface samples. With the exception of liquid whole egg and fresh bagged lettuce, which were tested in-house, all matrixes were tested by Marshfield Food Safety, Marshfield, WI, on behalf of Thermo Fisher Scientific. In addition, three matrixes (pork frankfurters, lettuce, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled laboratory study by the University of Guelph, Canada. No significant difference by probability of detection or McNemars Chi-squared statistical analysis was found between the candidate or reference methods for any of the food matrixes or environmental surface samples tested during the validation study. Inclusivity and exclusivity testing was conducted with 117 and 36 isolates, respectively, which demonstrated that the SureTect Salmonella species Assay was able to detect all the major groups of Salmonella enterica subspecies enterica (e.g., Typhimurium) and the less common subspecies of S. enterica (e.g., arizoniae) and the rarely encountered S. bongori. None of the exclusivity isolates analyzed were detected by the SureTect Salmonella species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation (enrichment time and temperature, and lysis temperature), which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.