RESUMO
Tektins are a highly conserved family of coiled-coil domain containing proteins known to play a role in structure, stability and function of cilia and flagella. Tektin proteins are thought to form filaments which run the length of the axoneme along the inner surface of the A tubule of each microtubule doublet. Phylogenetic analyses suggest that the tektin family arose via duplications from a single tektin gene in a unicellular organism giving rise to four and five tektin genes in bilaterians and in spiralians, respectively. Although tektins are found in most metazoans, little is known about their expression and function outside of a handful of model species. Here we present the first comprehensive study of tektin family gene expression in any animal system, in the spiralian annelid Platynereis dumerilii. This indirect developing species retains a full ancient spiralian complement of five tektin genes. We show that all five tektins are expressed almost exclusively in known ciliary structures following the expression of the motile cilia master regulator foxJ1. The three older bilaterian tektin-1, tektin-2, and tektin-4 genes, show a high degree of spatial and temporal co-regulation, while the spiralian specific tektin-3/5A and tektin-3/5B show a delay in onset of expression in every ciliary structure. In addition, tektin-3/5B transcripts show a restricted subcellular localization to the most apical region near the multiciliary arrays. The exact recapitulation of the sequence of expression and localization of the five tektins at different times during larval development indicates the cooption of a fixed regulatory and cellular program during the formation of each ciliary band and multiciliated cell type in this spiralian.
Assuntos
Cílios , Proteínas dos Microtúbulos , Animais , Filogenia , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismoRESUMO
BACKGROUND: Cilia and flagella are complex cellular structures thought to have first evolved in a last ciliated eukaryotic ancestor due to the conserved 9 + 2 microtubule doublet structure of the axoneme and associated proteins. The Tektin family of coiled-coil domain containing proteins was previously identified in cilia of organisms as diverse as green algae and sea urchin. While studies have shown that some Tektins are necessary for ciliary function, there has been no comprehensive phylogenetic survey of tektin genes. To fill this gap, we sampled tektin sequences broadly among metazoan and unicellular lineages in order to determine how the tektin gene complements evolved in over 100 different extant species. RESULTS: Using Bayesian and Maximum Likelihood analyses, we have ascertained with high confidence that all metazoan tektins arose from a single ancestral tektin gene in the last common ancestor of metazoans and choanoflagellates. Gene duplications gave rise to two tektin genes in the metazoan ancestor, and a subsequent expansion to three and four tektin genes in early bilaterian ancestors. While all four tektin genes remained highly conserved in most deuterostome and spiralian species surveyed, most tektin genes in ecdysozoans are highly derived with extensive gene loss in several lineages including nematodes and some crustaceans. In addition, while tektin-1, - 2, and - 4 have remained as single copy genes in most lineages, tektin-3/5 has been duplicated independently several times, notably at the base of the spiralian, vertebrate and hymenopteran (Ecdysozoa) clades. CONCLUSIONS: We provide a solid description of tektin evolution supporting one, two, three, and four ancestral tektin genes in a holozoan, metazoan, bilaterian, and nephrozoan ancestor, respectively. The isolated presence of tektin in a cryptophyte and a chlorophyte branch invokes events of horizontal gene transfer, and that the last common ciliated eukaryotic ancestor lacked a tektin gene. Reconstructing the evolutionary history of the tektin complement in each extant metazoan species enabled us to pinpoint lineage specific expansions and losses. Our analysis will help to direct future studies on Tektin function, and how gain and loss of tektin genes might have contributed to the evolution of various types of cilia and flagella.
Assuntos
Biodiversidade , Cílios/metabolismo , Proteínas dos Microtúbulos/classificação , Filogenia , Sequência de Aminoácidos , Animais , Células Eucarióticas/metabolismo , Evolução Molecular , Genoma , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/genéticaRESUMO
BACKGROUND: The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RESULTS: RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. CONCLUSIONS: Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Poliquetos/genética , Transcriptoma , Animais , Mapeamento Cromossômico , Análise por Conglomerados , Biologia Computacional/métodos , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Feminino , Ontologia Genética , Redes Reguladoras de Genes , Masculino , Anotação de Sequência Molecular , Fases de Leitura Aberta , FilogeniaRESUMO
Cultural confirmation following detection of a Listeria monocytogenespresumptive positive can take 3-7 days to finalize; this uncertainty is a point of frustration for food producers needing to make time-sensitive disposition decisions. To address the demand for shortened time-to-results, an alternative L. monocytogenes confirmation method consisting of two components, (i) a secondary screen using a different rapid method, and (ii) concurrent cultural isolation followed by next-day colony identification was evaluated. For the study, four food matrices (hot dogs, peanut butter, frozen vegetables, and multicomponent frozen meals) were inoculated with low levels (0.36-1.39 MPN/125 g) of L. monocytogenes per the AOAC guidelines for a matrix study. Analyses were performed on 125 g test portions and started with a PCR primary screen (Bio-Rad iQ-Check Listeria monocytogenes II). Next, all enriched food samples underwent a secondary screen by bioMérieux's GENE-UP LMO2 Real-Time PCR and VIDAS LMX ELFA along with streaking onto RAPID'L.mono Agar. Presumptive positive L. monocytogenes colonies were identified utilizing a high throughput rapid identification method (Hygiena's BAX System L. monocytogenes Real-Time PCR assay, Neogen's ANSR isothermal nucleic acid amplification assay, and Bruker's MALDI Biotyper). Importantly, this study evaluated multiple commercially available options for the secondary screen (n = 2) and rapid identification (n = 3) to allow for easy adoption by testing laboratories. Overall, there was no statistically significant difference (p ≤ 0.05) between the number of L. monocytogenes-positive 125 g samples obtained by the cultural reference method and the alternative confirmation methods (regardless of which method combinations were evaluated). Additionally, this study supports that, when both the primary and secondary screen methods yield a positive result, the sample could be considered a confirmed positive for L. monocytogenes.
Assuntos
Listeria monocytogenes , Listeria , Listeria monocytogenes/genética , Microbiologia de Alimentos , Alimentos , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: The Assurance® GDS for Cronobacter Tq II assay is a nucleic acid amplification system for the qualitative detection of Cronobacter. The method uses an upfront concentration of the target organism from the enrichment by immunomagnetic separation (IMS) using the PickPen® device. OBJECTIVE: The Assurance GDS for Cronobacter Tq II method was evaluated for Official Methods of AnalysisSM certification. METHOD: The matrix was compared to the ISO 22964:2017: Microbiology of the Food Chain-Horizontal Method for the Detection of Cronobacter spp. standard and using an alternative confirmation procedure. The alternative method was evaluated using 10 g test portions in an unpaired study design for powdered infant formula (milk based with iron and DHA) containing probiotics. Eleven technicians from eight laboratories located within the United States and Europe participated in the collaborative study. Statistical analysis was conducted according to the probability of detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence intervals across collaborators was calculated for each level between the candidate and reference method results and between the candidate presumptive and confirmed results. RESULTS: Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval of 0.03 (-0.18, 0.15). The dLPOD results indicate equivalence between the candidate method and reference method for the matrix evaluated. The method also demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. There were no false negative results; the false positive rate was determined and produced a value of <2%. CONCLUSIONS: Based on the data generated, the method demonstrated Assurance GDS for Cronobacter Tq II assay produced acceptable interlaboratory reproducibility data and statistical analysis. HIGHLIGHTS: The Assurance GDS for Cronobacter Tq II method is suitable for the rapid qualitative detection of Cronobacter in infant formulas, infant cereals, ingredients, and environmental samples.
Assuntos
Cronobacter , Microbiologia de Alimentos , Humanos , Lactente , Fórmulas Infantis/microbiologia , Grão Comestível , Cronobacter/genética , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Frequent testing for Legionella concentration in water is required by most health risk monitoring organizations worldwide. Domestic hot water and cooling tower water networks must be regularly controlled to prevent Legionnaires' disease, a potentially deadly lung infection. MICA Legionella is the fastest culture-based detection method for all serogroups of Legionella pneumophila, with automatic enumeration in 48 h and no need for confirmation. OBJECTIVE: This study compares the performance and robustness of MICA Legionella with the reference method ISO 11731:2017 for the enumeration of culturable L. pneumophila. METHODS: MICA Legionella and ISO 11731:2017 results were compared for domestic hot water and cooling tower water. Inclusivity and exclusivity were tested on reference and environmental strains. Ruggedness, lot-to-lot consistency, and stability of the reagents kit were also studied. RESULTS: Enumeration of L. pneumophila by MICA Legionella was statistically equivalent to ISO 11731:2017 in both matrixes. In cooling tower waters, MICA Legionella showed better sensitivity than ISO 11731:2017. It presented a 94% sensitivity and a 97% specificity. CONCLUSION: MICA Legionella is a highly sensitive and specific method for culturable L. pneumophila enumeration. It presents, in 48 hours, equivalent or better results than ISO 11731:2017. Its protocol is robust to variations. Its reagents kit is stable for up to 18 months. HIGHLIGHTS: MICA Legionella is a robust and reliable method for the enumeration of culturable L. pneumophila in domestic and cooling tower water. It reduces significantly the number of sample pretreatments required in ISO 11731:2017. Automatic identification and enumeration of L. pneumophila microcolonies eliminates the requirement to have skilled analysts and limits the results variability. It also greatly reduces the time to results to 48 h instead of 7-10 days with ISO 11731:2017 while providing statistically equivalent results.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Microbiologia da Água , Doença dos Legionários/prevenção & controle , ÁguaRESUMO
BACKGROUND: The AquaCHROM™ ECC method from CHROMagar is intended for the detection and enumeration of Escherichia coli and coliform bacteria in 100 mL water samples after 18-24 h of incubation at 35-37°C. OBJECTIVE: To validate the AquaCHROM ECC method for qualitative and quantitative detection of E. coli and coliforms with different water matrixes. METHODS: Inclusivity/exclusivity studies were conducted. AquaCHROM ECC was compared to U.S. cultural reference methods in unpaired matrix studies for detection of E. coli and coliforms in tap water, well water, lake water, and bottled water, and for enumeration in tap water, well water, and lake water. Three production lots of AquaCHROM ECC were tested for product consistency and stability. Variations in incubation time and temperature were evaluated in robustness testing. RESULTS: Inclusivity/exclusivity results demonstrated expected performance with the exception of three strains of Salmonella enterica, two species of Shigella, and one strain of Aeromonas, which turned the media blue or yellow. Results from the matrix studies demonstrated that AquaCHROM ECC and the reference methods are not statistically different for detection of E. coli and coliforms and statistically equivalent for enumeration of E. coli and coliforms. The AquaCHROM ECC powder production was proven to be consistent with a 24-month shelf life. Variation in temperature did not affect the method performance. Shortening the incubation time is not recommended. CONCLUSION: AquaCHROM ECC is an effective method for the detection and enumeration of E. coli and coliforms for the water matrixes evaluated. HIGHLIGHTS: The AquaCHROM ECC method is a quick, one-step method for the recovery and enumeration of E. coli and coliforms in 100 mL water samples. It is a non-agar-based chromogenic medium which provides a clear result without the use of a UV lamp.
Assuntos
Enterobacteriaceae , Escherichia coli , Água , Microbiologia de Alimentos , Água DoceRESUMO
BACKGROUND: The Clear Safety Listeria method utilizes polymerase chain reaction (PCR) amplification and targeted next-generation sequencing technology to detect Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. marthii, L. grayi, L. welshimeri, and L. seeligeri) in hot dogs and on selected environmental surfaces. OBJECTIVE: The aim was to validate the candidate method according to current AOAC guidelines. METHOD: The candidate method was compared to the reference method for hot dogs and the environmental surfaces. The method was also evaluated for inclusivity and exclusivity using 50 inclusivity strains and 30 exclusivity strains for each reported target. Product consistency and stability was tested and robustness was evaluated with changes in enrichment temperature, volume of sample treatment, and aliquot volume for PCR. RESULTS: The candidate method demonstrated no statistically significant differences using the probability of detection model between candidate and reference methods or between presumptive and confirmed results for all environmental surfaces and hot dogs. Additionally, the candidate method detected all inclusivity organisms and excluded all exclusivity organisms for each reported target. Product lots were shown to be consistent and data supported the kit's shelf life. Finally, the robustness study demonstrated no statistical differences when the volume of sample or the aliquot volume for PCR was altered. Increasing the incubation temperature to 37 ± 1 °C resulted in greater recovery of L. monocytogenes as compared to 35 ± 1 °C and 30 ± 1 °C. CONCLUSIONS: The Clear Safety Listeria method is statistically equivalent to the reference methods for the detection of L. monocytogenes and Listeria spp. in hot dogs and on selected environmental surfaces. HIGHLIGHTS: The Clear Safety Listeria method is an automated, highthroughput NGS-based method capable of detecting Listeria species in the hot dog and environmental samples within 28h.
Assuntos
Listeria monocytogenes , Listeria , Microbiologia de Alimentos , Listeria/genética , Listeria monocytogenes/genética , Reação em Cadeia da Polimerase , Aço InoxidávelRESUMO
BACKGROUND: The Thermo Scientific™ SureTect™ Listeria monocytogenes PCR Assay uses Solaris reagents for performing PCR for the rapid and specific detection of Listeria monocytogenes in a broad range of foods and selected environmental surfaces. OBJECTIVE: To demonstrate reproducibility of the SureTect Listeria monocytogenes PCR Assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g). To extend the scope of the method. METHOD: In the collaborative study, the candidate method was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, to extend the scope, six matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the Food Chain-Horizontal Method for the Detection and Enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection Method Reference Method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested. CONCLUSIONS: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination or false positive/negative data were reported, highlighting the ease of use, reproducibility, and robustness of the method.
Assuntos
COVID-19 , Listeria monocytogenes , Listeria , Microbiologia de Alimentos , Humanos , Listeria/genética , Listeria monocytogenes/genética , Pandemias , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Estados UnidosRESUMO
BACKGROUND: The Thermo Scientific™ SureTect™ Salmonella species PCR Assay utilizes Solaris™ reagents for performing PCR for the rapid and specific detection of Salmonella species in a broad range of foods and select environmental surfaces. OBJECTIVE: The aims were to demonstrate the reproducibility of the Thermo Scientific SureTect Salmonella species PCR Assay in a collaborative study using a challenging matrix, cocoa powder, and to extend the scope of the method. METHOD: In the collaborative study, the candidate method was compared to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Fourteen participants from nine laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection statistical model. In addition, 11 matrix studies were performed comparing the candidate method to the FDA/BAM Chapter 5 or US Department of Agriculture, Food Safety and Inspection Service, Microbiology Laboratory Guidebook 4.10 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Siluriformes (Fish) Products and Carcass and Environmental Sponges reference method. Nine of these matrices were also compared to the EN ISO 6579-1:2017/Amd.1:2020(E) Microbiology of the food chain-Horizontal method for the detection, enumeration and serotyping of Salmonella-Part 1: Detection of Salmonella spp.-AMENDMENT 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC reference method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False-positive and false-negative rates were determined for the matrix and produced values of <2%. The two PCR thermocyclers (QS5, 7500 Fast) performed equivalently. There were no result differences between candidate method confirmations and reference method confirmations. In the pre-collaborative matrix extension, the results from the matrix studies showed a comparable performance between the candidate method and the tested reference methods. CONCLUSIONS: Based on the data generated, the method demonstrated acceptable interlaboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 375 g cocoa powder is known to be a challenging matrix for PCR methods. No unusual cross-contamination or false-positive/negative was reported, highlighting the ease of use, reproducibility, and robustness of the method.
Assuntos
COVID-19 , Microbiologia de Alimentos , Animais , Humanos , Carne/análise , Pandemias , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , SARS-CoV-2 , Salmonella/genética , Estados UnidosRESUMO
BACKGROUND: The Thermo Scientific SureTect™ Listeria species PCR assay utilizes SolarisTM reagents for performing PCR for the rapid and specific detection of Listeria species in a broad range of foods and selected environmental surfaces. OBJECTIVE: To demonstrate reproducibility of the Thermo Scientific SureTect Listeria species PCR assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g), to extend the scope of the method. METHODS: In the collaborative study, the candidate method was compared to the US Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Ch. 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). The candidate method included its own confirmation procedure. Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, in order to extend the scope of the method, seven matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the food chain-Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection method reference method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested. CONCLUSION: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination, or false-positive/negative data was reported, highlighting the ease of use, reproducibility, and robustness of the candidate method.
Assuntos
COVID-19 , Listeria , Teste para COVID-19 , Microbiologia de Alimentos , Humanos , Listeria/genética , Pandemias , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Estados UnidosRESUMO
BACKGROUND: The Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR kits for the rapid detection of E. coli O157:H7 and non-E. coli O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, O145) from fresh raw spinach, fresh baby leaves, fresh cut tomatoes, frozen raw beef, raw beef trim, and beef carcass sponges. OBJECTIVE: Both assays were evaluated for AOAC®Performance Tested MethodsSM certification. METHODS: Detection and confirmation inclusivity/exclusivity, matrix, product consistency and stability, and robustness studies were conducted. In the matrix studies, the candidate method was validated against United States and international reference methods for STEC serotypes. RESULTS: Matrix studies showed no statistically significant differences between the candidate and reference method results when analyzed by probability of detection. For each inclusivity/exclusivity study, all inclusivity strains and no exclusivity strains were detected by either kit. Robustness testing demonstrated that the identification assay performed reliably despite method deviations; however, although not statistically significant, the screening assay performance was impacted. Product consistency and stability testing demonstrated no statistically significant differences between kit lots and storage time points. CONCLUSION: The data presented show that both assays constitute a rapid and reliable workflow for the detection and confirmation of E. coli O157:H7 and stipulated non-E. coli O157:H7 STEC serotypes from the tested matrixes. HIGHLIGHTS: Results are obtained in 80 min post-enrichment with both assays run simultaneously, allowing for the detection and confirmation of STEC within a single workflow.
Assuntos
Escherichia coli O157 , Escherichia coli Shiga Toxigênica , Solanum lycopersicum , Animais , Bovinos , Escherichia coli O157/genética , Microbiologia de Alimentos , Folhas de Planta , Reação em Cadeia da Polimerase em Tempo Real , Sorogrupo , Escherichia coli Shiga Toxigênica/genética , Spinacia oleracea , Estados UnidosRESUMO
BACKGROUND: The Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay is a real-time PCR assay for the detection of Staphylococcus aureus in dairy samples. OBJECTIVE: The Thermo Scientific SureTect Staphylococcus aureus PCR Assay was evaluated for AOAC Performance Tested MethodSM certification. METHODS: Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method's performance. For the matrix study, the method was validated on the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument and the Applied Biosystems 7500 Fast Real-Time PCR instrument against the ISO 6888-3:2003 Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species)-Part 3: Detection and MPN technique for low numbers, and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Ch. 12, Staphylococcus aureus, 2016, reference methods. RESULTS: Matrix studies showed no statistically significant differences between the candidate and reference methods or between presumptive and confirmed results. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant difference in assay performance after set method parameter deviations, and product consistency and stability studies demonstrated no statistically significant differences in performance between kit lots at different expiration points. CONCLUSION: The data presented show that the assay is a rapid and reliable workflow for the detection of S. aureus from dairy matrixes. HIGHLIGHTS: The PCR assay allows for fast, reliable detection of S. aureus in dairy matrixes with results obtained in as little as 80 min post enrichment.
Assuntos
Microbiologia de Alimentos , Staphylococcus aureus , Animais , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/genéticaRESUMO
BACKGROUND: The Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit is a real-time PCR assay for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. OBJECTIVE: The Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR Kit was evaluated for AOAC®Performance Tested MethodsSM certification. METHODS: Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method's performance. In the matrix studies, the method was validated against United States and international reference methods for Campylobacter detection. RESULTS: There were no statistically significant differences found in the matrix studies between the candidate and reference methods when analyzed by probability of detection. All 52 inclusivity strains and none of the 51 exclusivity strains tested were detected by the assay. Robustness testing demonstrated that the assay gave reliable performance with specific method deviations outside of the recommended parameters, and the real-time stability testing demonstrated that there were no statistically significant differences between kit lots, validating the stated shelf life of the kit. CONCLUSION: The data presented support the product claims that the Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR assay is suitable for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. HIGHLIGHTS: Presumptive results can be obtained in as little as 23 h. Microaerophilic incubators are not required for enrichment.
Assuntos
Campylobacter coli , Campylobacter jejuni , Campylobacter , Animais , Campylobacter/genética , Campylobacter coli/genética , Campylobacter jejuni/genética , Aves Domésticas , Produtos Avícolas , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
BACKGROUND: The Clear Safety Salmonella method was modified to improve sample preparation, PCR reagents, library preparation, flow cell quality control, library loading mix, priming mix, and sequencing kit reagents and steps. OBJECTIVE: To evaluate the modified Clear Safety Salmonella method (manual and automated) via independent and method developer validation studies according to current AOAC INTERNATIONAL Validation Guidelines. METHOD: Performance of the modified Clear Safety Salmonella method (manual and automated) was assessed for selectivity (using 105 inclusive and 30 exclusive strains), probability of detection in matrixes, product consistency, stability, and robustness. The modified Clear Safety Salmonella method was compared with the appropriate reference method for Salmonella detection on 4 inch × 4 inch stainless steel environmental surfaces, and in chicken carcass rinse (30 mL), raw ground chicken (375 g), dry pet food (375 g), and ready-to-eat deli turkey breast (375 g). RESULTS: The modified Clear Safety Salmonella method (manual and automated) demonstrated no statistically significant differences between the candidate and reference method probability of detection or between the presumptive and confirmed results for all target food matrixes and the stainless steel surface. Additionally, the modified method (manual and automated) detected all 105 inclusivity organisms and excluded all 30 exclusivity organisms. The product consistency and kit stability studies showed no statistical differences between lots or over the term of the kit's shelf life. In robustness studies, changes in enrichment time, diluted sample volume, and sample volume for PCR did not show any statistical difference in terms of assay performance. CONCLUSIONS: The modified Clear Safety Salmonella method (both manual and automated) is statistically equivalent to or better than the reference methods. HIGHLIGHTS: The Clear Safety Salmonella method utilizes PCR amplification and targeted next-generation sequencing technology to selectively detect Salmonella enterica.
Assuntos
Salmonella enterica , Aço Inoxidável , Animais , Microbiologia de Alimentos , Aves Domésticas , SalmonellaRESUMO
BACKGROUND: The PathogenDx family of assays uses microarray technology to simultaneously detect the presence of bacterial and fungal pathogens in food products, environmental surfaces, and cannabis products. OBJECTIVE: The Detectx Combined assay was validated for the detection of Aspergillus, (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus), Salmonella, and a broad range of STEC (stx1 and/or 2) species. The validation consisted of two matrix studies in dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol) flower, product consistency, stability, robustness, and inclusivity and exclusivity for two targets: Aspergillus and STEC. METHOD: The PathogenDx Detectx Combined assay was evaluated with 30 replicates in each matrix and confirmed according to the instructions outlined in this study. RESULTS: Results of the validation study met the requirements of AOAC Standard Method Performance Requirement (SMPR®) 2020.002 and 2020.012. In the inclusivity and exclusivity study, all target isolates (Aspergillus and STEC) were correctly detected. For the exclusivity study, 26 out of 30 Aspergillus and 30 out of 30 STEC non-target strains were correctly excluded. In the matrix study, the PathogenDx Detectx Combined assay showed no significant statistical differences between confirmed results for dried hemp and cannabis flower. Robustness testing indicated that small changes to the method parameters did not impact the performance of the assay. Stability and consistency studies verified that the assay's shelf-life claims were appropriate, and manufacturing of the assay was consistent. CONCLUSIONS: The validation study indicated that the PathogenDx DetectX Combined assay was successful in detection of the new target analytes (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus and STEC containing stx1 and/or 2) and could successfully recover these organisms and Salmonella from dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol). HIGHLIGHTS: The PathogenDx DetectX Combined Assay will be the first PTM approved multiplex assay for Aspergillus, E. coli and Salmonella that does not require an enrichment step.
Assuntos
Cannabis , Escherichia coli Shiga Toxigênica , Aspergillus , Dronabinol , Flores , Microbiologia de Alimentos , SalmonellaRESUMO
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.
Assuntos
Microbiologia de Alimentos , Salmonella , Animais , Técnicas Bacteriológicas , Galinhas , Aço InoxidávelRESUMO
BACKGROUND: The Solus One Salmonella immunoassay utilizes Salmonella specific selective media and automated liquid handling, for the rapid and specific detection of Salmonella species in select food types. OBJECTIVE: The candidate method was evaluated using 375 g test portions in an unpaired study design for a single matrix, instant non-fat dry milk (NFDM) powder. METHOD: The matrix was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 5 Salmonella reference method. Eleven participants from 10 laboratories within academia and industry, located within the United States, Mexico, South Africa, Germany, and the United Kingdom, contributed data for the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level [0 colony forming units (CFU)/test portion], a low inoculum level (0.2-2 CFU/test portion) and a high inoculum level (2-5 CFU/test portion). Statistical analysis was conducted according to the Probability of Detection (POD) statistical model. RESULTS: Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval between the candidate method confirmed (both alternative and conventional confirmation procedures) and the reference method of 0.07 (-0.02, 0.15). CONCLUSIONS: The dLPOD results indicate equivalence between the candidate method and the reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for the matrix and produce values of <2%. HIGHLIGHTS: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.
Assuntos
Microbiologia de Alimentos , Salmonella , Alemanha , Humanos , Reprodutibilidade dos Testes , Salmonella/isolamento & purificação , Reino Unido , Estados UnidosRESUMO
BACKGROUND: The MC-Media Pad® Rapid Aerobic Count (RAC) is a ready-to-use culture device combining a test pad coated with medium and water absorption polymers that are designed for the rapid quantification of total aerobic bacteria in food products. OBJECTIVE: The MC-Media Pad RAC was compared to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, Chapter 3.02: Quantitative Analysis of Bacteria in Foods as Sanitary Indicators for raw ground pork and the Standard Methods for the Examination of Dairy Products, Chapter 6: Microbial Count Methods for yogurt drink. METHOD: The candidate method was evaluated against the reference methods using a paired study design in a multi-collaborator study, following the current AOAC INTERNATIONAL Official Methods of AnalysisSM Appendix J guidelines. Three target contamination levels (low, medium, and high) were evaluated. MC-Media Pad RAC devices were enumerated after 24 and 48 h of incubation. RESULTS: Plate counts obtained by both methods were log10-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and reproducibility SD were determined for each contamination level. All 95% confidence intervals for mean difference fell easily within ±0.10, the performance requirement being ±0.5. CONCLUSION: The MC-Media Pad RAC (for both 24 and 48 h) and both reference methods for each contamination level were therefore shown to be equivalent, with 97.5% confidence. HIGHLIGHTS: The new method offers a convenient alternative to the reference methods for detection of aerobic plate count in food products, yielding reliable and comparable results in 24 or 48 h compared to 48 h for the reference methods.
Assuntos
Bactérias Aeróbias , Microbiologia de Alimentos , Contagem de Colônia Microbiana , Meios de Cultura , Reprodutibilidade dos Testes , IogurteRESUMO
Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30â¯mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23â¯mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24â¯h in the modified (BPWâ¯+â¯L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.