Evaluation of PhageDX Salmonella Assay for Salmonella Detection in Hydroponic Curly Lettuce

Lettuce is one of the most consumed leafy vegetables worldwide and has been involved in multiple foodborne outbreaks. Salmonella is one of the most prevalent etiological agents of foodborne disease (FBD) in lettuces, and its detection may take several days depending on the chosen method. This study evaluates a new rapid method that uses recombinant bacteriophages to detect Salmonella in hydroponic curly lettuce. First, the ability of the assay to detect six Salmonella serovars at three different concentrations (1, 10, and 100 CFU/well) was tested. Second, the detection of Salmonella was tested in lettuces using a cocktail of the same Salmonella serovars and concentrations after a 7 h enrichment. The results of these experiments showed that the detection limit was dependent on the serovar tested. Most serovars were detected in only 2 h when the concentration was 100 CFU/well. Salmonella was detected in 9 h (7 h enrichment + 2 h bioluminescence assay) in all lettuce samples with 10 CFU/25 g or more. Salmonella detection was not influenced by natural microbiota of lettuces. This study demonstrated that the phage assay was sensitive and faster than other detection methods, indicating that it is a better alternative for Salmonella detection on lettuces.

The Validation of the MEMP Salmonella Assay: AOAC Performance Tested MethodsSM 042002

Background: The Molecular Environmental Monitoring Program (MEMP) Salmonella Assay is a quick and reliable method for detecting Salmonella species in environmental samples. The assay incorporates a real-time PCR approach to identifying Salmonella cells expressed from the swab sample. The assay does not require an enrichment step, leading to much faster time to a negative result. Objective: This report details the method validation study to validate the MEMP using environmental surface swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Methods: Matrix studies, inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted to assess the method’s performance. In the matrix studies, this method was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Chapter 5 for environmental surface sponges and swabs. Results: Inclusivity/exclusivity testing showed that this assay was able to detect all 100 Salmonella strains tested while excluding the 30 non-Salmonella species. There were no statistically significant differences found between the candidate and reference methods. Small variations in critical test parameters (volume of extraction reagent and volume of extracted DNA sample) did not adversely affect the assay’s performance, and stability testing indicated consistent results for at least one year. Conclusions: The data presented in this report show that the candidate method performed as well as the reference method; therefore, it can be used in place of the reference method for detecting Salmonella species. Highlights: The MEMP Salmonella Assay is the first and only AOAC PTM approved method for detecting Salmonella on surfaces without enrichment.

Cancer interception by interceptor molecules: mechanistic, preclinical and human translational studies with chlorophylls

Before ‘cancer interception’ was first advocated, ‘interceptor molecules’ had been conceived as a sub-category of preventive agents that interfered with the earliest initiation steps in carcinogenesis. Three decades ago, a seminal review cataloged over fifty synthetic agents and natural products that were known or putative interceptor molecules. Chlorophylls and their derivatives garnered much interest based on the potent antimutagenic activity in the Salmonella assay, and the subsequent mechanistic work that provided proof-of-concept for direct molecular complexes with planar aromatic carcinogens. As the ‘interceptor molecule’ hypothesis evolved, mechanistic experiments and preclinical studies supported the view that chlorophylls can interact with environmental heterocyclic amines, aflatoxins, and polycyclic aromatic hydrocarbons to limit their uptake and bioavailability in vivo. Support also came from human translational studies involving ultralow dose detection in healthy volunteers, as well as intervention in at-risk subjects. Antimutagenic and antigenotoxic effects of natural and synthetic chlorophylls against small alkylating agents also highlighted the fact that non-interceptor mechanisms existed. This gave impetus to investigations broadly related to free radical scavenging, anti-inflammatory effects, immune modulation and photodynamic therapy. Therapeutic aspects of chlorophylls also were investigated, with evidence for cell cycle arrest and apoptosis in human cancer cells. As the science has evolved, new mechanistic leads continue to support the use and development of chlorophylls and their porphyrin derivatives for cancer interception, beyond the initial interest as interceptor molecules.

The Validation of the SIMUL-qPCR Salmonella Assay for AOAC Research Institute Performance Tested MethodsSM Certification (Certificate No. 042001)

Background The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Salmonella Assay is a quick, reliable method for detecting Salmonella species in environmental and food samples. The assay multiplexes several targets in one run to identify Salmonella species. For most matrixes, the assay uses buffered peptone water for enrichment purposes. However, the assay can be used in conjunction with the SIMUL-qPCR Top 7 STEC Assay for raw beef trim and raw ground beef when proprietary media has been used. This media is optimized for single-step enrichment and recovery of Enterohemorrhagic E. coli and Salmonella in those matrixes. Objective This report details the method validation study to validate raw beef trim, raw ground beef, fresh raw ground poultry, ready-to-eat cooked poultry, dry pet food, pasteurized liquid eggs, peanut butter, frankfurter/sausage, poultry carcass rinse, and environmental surface sponges and swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Methods Matrix studies, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method’s performance. Results Inclusivity/exclusivity testing showed the SIMUL-qPCR Salmonella Assay was able to detect Salmonella strains while excluding the non-Salmonella isolates. There were no statistically significant differences found between the candidate and reference methods in the matrix studies. Small variations in critical test parameters (enrichment time, extraction reagent volume, and extracted sample volume) didn’t adversely affect the assay’s performance, and stability testing indicated consistent results for at least one year. Conclusions The data presented demonstrate the SIMUL-qPCR Salmonella Assay is a reliable method for detecting Salmonella.

ANTIMUTAGENIC POTENTIAL OF LIV52 BY AMES SALMONELLA/MAMMALIAN: MICROSOME MUTAGENICITY ASSAY

ABSTRACTObjective: Liv52, a nontoxic herbal preparation is reported to be clinically active in hepatotoxicity and a wide range of hepatic disorders. This studywas undertaken to evaluate the mutagenic/antimutagenic potential of Liv52 using Salmonella mutagenicity test.Methods: Ames Salmonella/Mammalian - microsome mutagenicity test was used to evaluate the mutagenic or antimutagenic potential of Liv52.Salmonella typhimurium tester strains TA1537, TA1538, TA100, and TA102 were used for mutagenicity testing. The antimutagenicity study wascarried out in tester strains TA1538 and TA100 against various standard mutagens with and without metabolic activation.Results: Liv52 did not show any mutagenic potential both with and without metabolic activation, whereas in TA1538 and TA 100 tester strains, Liv52showed 48.4% and 47.2% of inhibition of his+ revertants induced by 4-nitro-O-phenylenediamine, respectively. Further with metabolic activationin TA1538, Liv52 showed 99.8%, 99.8%, and 100% inhibition of his+ revertants induced by 2-aminofluorene, 2-anthranine, and cigarette smokecondensate, respectively. In TA100 maximum of 100%, 100%, 97.7%, and 100% inhibition of his+ revertants induced by 2-aminofluorene, 2-anthranine,benzo(a)pyrene, and cigarette smoke condensate, respectively, were observed. A significant enhancement of inhibition of his+ revertants induced byall the above said mutagens were observed in preincubation modification method.Conclusion: Liv52 was found to be nonmutagenic in Salmonella assay, whereas manifested the antimutagenic potential both with and withoutmetabolic activation. The enhanced antimutagenic activity of Liv52 on preincubation indicates that the antimutagenic factor(s) may be desmutagenicin nature. The exact mechanism by which Liv52 exerts antimutagenic potential is not known. The possibility of having diverse antimutagenic factorsin Liv52 which act by different mechanisms are strongly indicated.Keywords: Liv52 syrup, Salmonella mutagenicity test, Ames test, Desmutagens.

Validation of FoodChek™ - Salmonella for Rapid Detection of Salmonella in Eggs, Derivative Products, and the Environment

The FoodChek™ - Salmonella assay is an immunomagnetic lateral flow assay for the rapid detection (shorter than 24 h) of the most frequently isolated Salmonella (groups B–E) in eggs, egg-derivative products, and environmental surfaces. The FoodChek - Salmonella assay correctly identified 99.6% (239/240) of the samples tested in the matrix studied, and the statistical analysis of the method comparison study results demonstrated that it performs as well as U.S. culture-based reference methods. Ninety-nine percent of the 103 Salmonella strains tested belonging to serogroups B–E were detected during the inclusivity study. Concerning the exclusivity, 31 nontarget strains were tested. No cross-reactivity was observed in FoodChek - Salmonella assay enrichment conditions. In addition, the assay shows strong robustness, good stability, and consistency among lots. The present study proves that the assay is an effective tool for the rapid detection of Salmonella spp. in whole liquid eggs, liquid egg white (liquid egg albumen), shell eggs, dried whole eggs, dried egg yolks, and environmental surfaces as stainless steel, plastic, rubber, ceramic tiles, and sealed concrete.

DNAble® Molecular Detection Kit for Salmonella

The DNAble Salmonella detection assay utilizes single overnight culture enrichment, user-friendly sample preparation, and isothermal DNA amplification for Salmonella detection. This report describes studies performed in support of AOAC Research Institute Performance Tested MethodSM certification of the DNAble assay. Selectivity (inclusivity and exclusivity) studies were performed in the sponsor's laboratory. DNAble detected 119 out of 120 Salmonella isolates, representing 100 Salmonella serovars, in the inclusivity study while none of the 35 diverse non-Salmonella strains (32 species) tested was detected in the exclusivity study. Consistency (lot-to-lot and stability), instrument variation, and robustness studies were also conducted by the sponsor. Statistically equivalent assay performance was observed in these studies demonstrating robust assay manufacture and performance despite variation of multiple parameters in these challenges. Matrix studies, performed in an independent laboratory, evaluated DNAble assay performance in dry pet food, on stainless steel surfaces, and poultry environmental drag swab samples. Two sample sizes (25 and 375 g) and two culture volumes (9:1 and 3:1, v/w) were evaluated in separate matrix studies for dry pet food to provide multiple certified testing options for assay users. DNAble assay performance for dry pet food and stainless steel was compared to the procedures described in the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual, Chapter 5, Salmonella. Assay performance for drag swabs was compared to protocols dictated in the FDA Environmental Sampling and Detection of Salmonella in Poultry Houses guidelines. Matrix study results demonstrated statistically equivalent DNAble assay performance compared to these reference methods, ensuring that the DNAble assay provides results comparable to those of the reference methods.

Evaluation of the ANSR® for Salmonella Assay for Identification of Salmonella spp. from Colony Picks from Selective/Differential Agar Media: First Action 2013.14

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.