Review of Salmonella detection and identification methods: Aspects of rapid emergency response and food safety

Salmonella has been recognized as a major and important foodborne pathogen for humans and animals over more than a century, causing human foodborne illness as well as high medical and economical cost. Accordingly, the effort to develop efficient and reliable Salmonella detection methods continues. This paper reviews and describes the development and application of commercially available Salmonella detection methods. These are categorized into several groups based on the principle applied: conventional culture methods, immunology-based assays, nucleic acid-based assays, miniaturized biochemical assays, and biosensors. Conventional culture methods serve as the basis in food testing laboratories despite rather laborious and time-consuming protocols. Considerable progress in rapid methods using emerging technologies yield faster answers and higher throughput of samples. This paper also shows and analyzes Salmonella test results and summarizes the features and limitations of the studies involving Salmonella detection methods developed for emergency response, mainly by food emergency response laboratories participating during recent fiscal years. The emergency response laboratories utilize Salmonella detection methods possessing properties that include simplicity, versatility, high sensitivity, good specificity, and cost efficiency. Collaboration of the food emergency response laboratories in the development of these technologies is important essentially to compare for the purpose of continually improving Salmonella detection methods.     

Incidence of citrinin in red yeast rice and various commercial Monascus products in Taiwan from 2009 to 2012

Red yeast rice, produced by the fermentation of red yeast (Monascus purpureus) with white rice, has long been used in food colouring and meat preservation in Asia. Recently, powdered red yeast rice and its formulated products, used worldwide as dietary supplements, have the ability of reducing blood-lipid levels in humans. However, some Monascus strains could produce the mycotoxin citrinin as a secondary toxic metabolite so that commercial Monascus products are of a serious concern to the public now. The aim of this study was to obtain information about the occurrence of citrinin in red yeast rice and various commercial Monascus products in Taiwan from 2009 to 2012. A simple and rapid HPLC-fluorescence method was developed to detect citrinin in red yeast rice and Monascus products. The method's performance was acceptable in terms of recoveries, which ranged from 81.2% to 94.3% for citrinin-spiked samples at levels of 0.1, 1 and 10 mg/kg, and the relative standard deviation ranged from 2.5% to 5.7%. The limit of quantification of 0.05 mg/kg was achieved. The survey results showed that among total 302 samples, the incidences of citrinin contamination were 69.0%, 35.1% and 5.7% for red yeast rice (raw material), dietary supplements and red yeast rice processed products, respectively. The mean contamination levels were 13.3, 1.2 and 0.1 mg/kg for red yeast rice (raw material), dietary supplements and red yeast rice processed products, respectively. Such high citrinin contamination rate and level is worthy of note.     

REACTION KINETICS FOR OLEFIN HYDROGENATION OVER COBALT-MOLYBDENUM HYDROTREATING CATALYST AND MODEL STUDY OF CATALYST PROPERTIES

ABSTRACT

Over the years, several analytical methods have been applied to various heavy crude oil residua and their processed products to understand the chemistry behind residuum upgrading processes. The ultimate aim has been to predict prccessability of specific feeds. However, few, if any analytical methods have been found which adequately perform this task. This paper examines selected processing experiments by the following techniques – clusion chromatography with element specific detection, D 2007-80 with as-phaltene precipitation (SARA) separation, hydrogen distribution and incorporation by NMR – cusses whether the analytical technique has any potential to predict prccessability

From the size exclusion chromatography with element specific detection studies, an intimate relationship appears between the catalyst pore size and molecule size based on examination of the size behavior of feeds and pilot-plant products. From the D 2007-80 and asphaltene separations, the quality of asphaltenes appears to be related to the relative ease of processability of at least two different feeds. From the hydrogen distribution studies, hydrogen utilization was found to be feed dependent and could be directed by processing type. All these trends have some potential towards the formulation of a residuum processability scheme, however no method is as yet globally satisfactory.     

Application of in situ loop-mediated isothermal amplification method for detection of Salmonella in foods

In situ loop-mediated isothermal amplification (in situ LAMP) as a novel technique for detection of food-borne pathogens, has been successfully applied to detect Salmonella in artificially contaminated eggshells. Escherichia coli C600 was used as a negative control in specificity experiment. The sensitivity of the in situ LAMP assay was confirmed by the test in serial 10-fold dilutions of Salmonella cells, and the detection limit of this method may reach 1 CFU/cm² on eggshells. Compared with traditional culture methods and PCR-based methods, the in situ LAMP assay is advantageous on rapidity, high specificity, less time consumption and ease in operation. This is the first study carried out to apply the in situ LAMP method in practical food-borne pathogens detection. The results indicate that this method has great potential for use in food safety laboratories.     

Importance of acetic acid bacteria in food industry

Acetic acid bacteria (AAB) comprise a group of gram-negative or gram-variable, ellipsoidal to rod-shaped cells that have an obligate aerobic metabolism with oxygen as the terminal electron acceptor. In the first classification of AAB, two main genera were determined as Acetobacter and Gluconobacter, but nowadays twelve genera are recognized and accommodated to the family Acetobacteraceae, the Alphaproteobacteria: Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swaminathania, Saccharibacter, Neoasaia, Granulibacter, Tanticharoenia and Ameyamaea. Isolation, purification, identification and preservation of AAB are very difficult. Phenotypic methods based on physiological abilitiesies have been used for identification of AAB by using various media. These phenotypic properties have now been complemented or replaced by molecular techniques, which are DNA and RNA based techniques.AAB are widespread in nature on various plants (fruits, cereals, herbs, etc.). They are important microorganisms in food industry because of their ability to oxidize many types of sugars and alcohols to organic acids as end products during fermentation process. The best known industrial application of AAB is vinegar production. This group of bacteria is also used in cellulose and sorbose production. On the other hand, the oxidizing ability of AAB could have spoilage effect in some products such as in wine. The aim of the present review is to introduce the importance of AAB in food industry by showing their current taxonomy, enumeration, isolation and identification methods, isolation sources and beneficial effects in food production systems.     

Rapid and specific detection of Listeria monocytogenes in smoked salmon with BAX®-PCR

Members of the genus Listeria are ubiquitous, and are therefore also common to the food and the environment. Among them, only Listeria monocytogenes has a pathogenic potential, and can cause infectious diseases (listeriosis) in humans. Conventional microbiological testing methods are labour-intensive and time consuming (4–5 days), and often require a number of different culture media for final isolation and confirmatory tests. In order to overcome these limitations, numerous rapid methods have been developed in recent years. DNA-based methods such as the polymerase chain reaction (PCR) have increasingly been used for rapid and sensitive detection of L. monocytogenes. Among the various available PCR assays, we used the BAX^® system (with two different detection procedures: gel detection and automated detection) to screen for L. monocytogenes in samples of vacuum packaged cold smoked salmon. A total of 27 samples were used for this study. The method was compared to the German standard microbiological detection method according to DIN 11290-1 and -2. Detection of Listeria and L. monocytogenes from salmon samples was performed using Palcam enrichment medium, followed by plating on both Palcam agar and ALOA agar. The BAX^® assay gave identical results for 26 food samples compared to the standard method, including 15 positives. Only in one case the BAX^® system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. In naturally contaminated food samples, the BAX^® method gave good results after 24–48 h. Application of this rapid method is simple and time saving.     

Casamino Acids and Oxyrase enhance growth of Listeria monocytogenes in multi-pathogen enrichments

Rapid methods have been developed as relatively faster alternatives to plate culture for the detection of pathogenic bacteria in foods. However, since most rapid methods are subject to logistical limitations (e.g., sample volume size, analysis time, matrix effects) and/or a detection scheme with insufficient sensitivity needed to detect very low levels of bacteria in foods, culture enrichment is often employed to increase the concentration of targeted pathogens prior to detection. Multiplexed rapid detection platforms, capable of simultaneous detection of different bacteria in a single sample, necessitate co-enrichment (or mixed culture enrichment) of as many different targeted microorganisms as possible in a timely manner. This investigation compares the growth of four major foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica) inoculated into pristine media or ground pork and enriched in various culture media. Initial results revealed that, after 24 h incubation, the growth of L. monocytogenes (the slowest-growing pathogen examined) was increased by approximately 1-log by the supplementation of Universal Preenrichment Broth with Casamino Acids and/or Oxyrase. Overnight (24 h) growth of L. monocytogenes in ground pork enrichment cultures was enhanced up to ca. 2-log by the addition of either Casamino Acids or Casamino Acids and Oxyrase for each of the tested growth media. Ultimately, an overnight culture of the inoculated pathogens in any of the selected media containing both Casamino Acids and Oxyrase was observed to yield target bacterial concentrations that were at sufficient levels (between 10e5 and 10e6 CFU/mL) for detection by most rapid methods.     

PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products

Alternaria spp. contamination and subsequent production of mycotoxins is a common problem in vegetable crops. Identification of Alternaria species by traditional methods requires specific skills and may not detect toxigenic moulds inactivated by food processing. By using molecular methods such as PCR the detection of Alternaria spp. becomes possible directly from the food or feed samples. In this study, a PCR method based on the Internal Transcribed Spacer (ITS) genetic marker has been used for detection of Alternaria spp. in raw and processed commercial tomato samples. Occurrence of altenuene, alternariol and alternariol methyl ether in the samples was analysed by high-performance liquid chromatography (HPLC) in order to assess the ability of the PCR assay to identify tomato samples containing Alternaria mycotoxins. The PCR assay revealed the presence of Alternaria spp. DNA in 41 out of 90 commercial samples (45.6%), while HPLC detected at least one of the Alternaria mycotoxins within 31 of the PCR positive samples. Detection of Alternaria DNA correlated well with the presence of the analysed Alternaria mycotoxins, indicating that the PCR protocol developed in this work for detection of Alternaria spp. DNA could be used as an indirect marker of the presence of Alternaria mycotoxins in raw and processed tomato products.     

Verifying the red wines adulteration through isotopic and chromatographic investigations coupled with multivariate statistic interpretation of the data

The assessment of wine traceability and authenticity is a major concern that has gained a lot of interest internationally since the wine has always been subjected to various fraudulent practices. Practiced since ancient times, wine fraud has become more sophisticated in the present day, taking many forms. Consumers, regulatory bodies and manufacturers are all interested to have reliable analytical tools and information to allow the authentication and detection of wine adulteration or incorrect labelling. This research study evaluates and proposes a possible strategy for the detection of adulterated sweet or medium sweet red table wines using appropriate chemical parameters which can reveal prohibited practices in the winemaking process. The work is performed on 29 table wine samples, bought from the market, packed in PET bottles. Exogenous addition of sugar and water in the counterfeited table red wines was detected by the measurement of stable isotopes content (δ¹³C and δ¹⁸O) known as origin markers and supplementary confirmed by other classical parameters, as the alcoholic strength of the wines (‰ vol.) and the presence of 5-(hydroxymethyl)-2-furaldehyde (HMF) and of synthetic sweeteners or synthetic red dyes used to correct deficiencies of taste and colour. Additionally, the nature and profile of anthocyanins, as indicator of the red colour of wine, was investigated in the table wines and then compared with that of authentic wines obtained by microvinification of Vitis vinifera, in order to determine their authenticity.     

Fast determination of α- and β-thujone in alcoholic beverages using solid-phase extraction and gas chromatography

Determination of α- and β-thujone concentration in various foodstuffs is of great importance due to their antagonistic action against GABA receptors. The present study describes a fast, simple and sensitive method of α- and β-thujone analysis in alcoholic beverages using gas chromatography combined with solid phase extraction (SPE). The developed method is characterized by: high yield (above 98%); and low sufficient detection limit 0.033 mg/L; high precision (RSD below 1.8%); excellent linearity for α- and β-thujone performed in the concentration range 5.0-300.0 mg/L for α-thujone and 0.5-30.0 mg/L for β-thujone with R² = 0.9995 and R² = 0.9992, respectively. The presented analytical approach constitutes substantial improvement on the previously reported methods, which involve solvent consuming liquid-liquid extraction or less selective and difficult to calibrate solid phase micro-extraction (SPME).     

The food safety impact of the Codex sampling plans for Listeria monocytogenes in ready-to-eat foods: An empirical case study applying the FAO/WHO sampling plan tool

Sampling plans are specified by the Codex Alimentarius Commission microbiological criteria for Listeria monocytogenes in ready-to-eat foods. This case study evaluates the direct food safety impact of the Codex sampling plans as estimated by the FAO/WHO web-based microbiological sampling plan analysis tool under different assumptions about the pathogen distribution, test procedures, and the fraction of lots tested. The case study uses L. monocytogenes concentration data available for deli-type salads to empirically illustrate application of the sampling tool. The results indicate that the estimated impact of the sampling plans is dependent on the partitioning of total observed variance into its within- and between-lot components. The presence-absence based sampling plan is relatively insensitive to the substantial uncertainty and variability of the sensitivity of the reference method for detection of L. monocytogenes. The analytical sample size for enumeration impacts the ability of the concentration-based sampling plan to discriminate between compliant and non-compliant lots. Reducing the frequency of lot testing dramatically changes the statistical properties of the sampling schemes. Skip-lot sampling places greater importance on compliance assurance than on the direct, curative impact of lot acceptance sampling.     

Shiga toxin-producing Escherichia coli in food: Incidence,ecology, and detection strategies

Commensal Escherichia coli are commonly utilized for investigating the genetic and biochemical requirements of microorganisms, and have served in a wide variety of applications. Pathogenic E. coli known as Shiga toxin (Stx)-producing E. coli (STEC) are associated with various food products including ground beef. These pathogens are present in a wide range of environments, and have caused numerous foodborne outbreaks and recalls. These outbreaks and the increased awareness of STEC have led to certain STEC serotypes to be declared adulterants in non-intact raw meat. Various STEC detection methods have been investigated, and numerous cultural and molecular-based detection methods continue to be modified to meet regulatory requirements. However, STEC serotypes may possess certain characteristics that lead to bias in the likelihood of a certain serotype being detected in an assay. Understanding the characteristics of these STEC serotypes will provide means for optimizing the detection platforms, and as a result limit foodborne illness and recalls caused by STEC due to enhanced cultural and molecular detection capabilities.     

A PCR protocol using inl gene as a target for specific detection of Listeria monocytogenes

Polymerase chain reaction is a powerful technique for detection of pathogens in foods. It is a rapid procedure with both sensitivity and specificity for quick detection and identification of specific pathogenic bacteria from different sources. Listeria monocytogenes detection methods based on PCR amplification of the iap, prfA and hly gene sequences have been reported. The present study undertakes the development of an alternative PCR method using the inl gene sequences as a target to detect pathogenic L. monocytogenes. The presence of a unique and specific DNA amplification fragment of 760 bp for the intragenic repeats B of the inlA gene in all strains of L. monocytogenes as compared to none in other Listeria and unrelated Gram positive and Gram negative species confirms that this procedure is an alternative PCR protocol for detection of L. monocytogenes.     

Detection of foodborne bacterial zoonoses by fluorescence in situ hybridization

The efficient and timely detection of bacterial pathogens remains a major public health concern throughout the world. Fluorescence in situ hybridization (FISH) is a promising tool to detect bacteria since it incorporates the advantages of rapid detection methods with the live/dead differentiation capacity of the gold standard culture methods. However, multiplexing pathogen detection, weak FISH signals and the establishment of a quantitative and sensitive direct enumeration approach remain troublesome obstacles for a widespread use in food microbiology. Therefore, we developed and tested a comprehensive set of highly specific multiplex-FISH tests for the simultaneous detection of various foodborne bacterial zoonoses, including important pathogens like Salmonella enterica, thermophilic Campylobacter and Listeria monocytogenes. The detection of thermophilic Campylobacter spp., the most frequent bacterial zoonosis in the EU, in artificially spiked chicken breast by FISH proved to be as sensitive as the conventional ISO standard, but results were available much earlier. Strongly enhanced FISH signals for Campylobacter spp., enabling detection in matrices with high background fluorescence, were accomplished by employing several probes for this target group. For the direct detection of bacteria, independent of cultural enrichment, filtration proved to be appropriate although this method is less sensitive and thus primarily suitable for higher bacterial loads. The type of membrane filter as well as the fluorescence channel significantly influenced the efficiency of detection. Furthermore, the implementation of GFP-expressing bacteria as a quantitative standard allowed the enumeration of target pathogens after filtration. In summary, our results demonstrate the applicability of FISH for food microbiology and offer new solutions for prevalent problems in FISH-testing.     

Rapid method based on immunoassay for determination of paraquat residues in wheat,barley and potato

The detection of bipyridine herbicides residues in food samples is hampered due to their particular physico-chemical features, which requires the application of specific extraction and analytical procedures, which disqualifies them from being incorporated into the multi-residue methods (MRMs). There is a need for alternative robust and efficient analytical screening methods, and in this respect, we present here a fast and reliable immunochemical analytical procedure for the detection of paraquat (PQ) residues in food samples, particularly potato, barley and wheat. The procedure involves the extraction with 1 N HCl:MeOH at 80 °C, followed by centrifugation and filtration, and the extracts can be directly measured by a microplate-based ELISA without any other sample treatment or clean-up, except from buffering the solution and adjusting the pH. Selective polyclonal antibodies, were raised against N-(4-carboxypent-1-yl)-N′-methyl bipyridilium acid (hapten PQ1), and used to establish a high sensitive immunochemical analytical assay, able to measure simultaneously many samples. Under these conditions the accuracy is very good, with almost quantitative recoveries. The non-specific interferences caused by the matrix are negligible for the case of potato and wheat, while for barley it is necessary to further dilute the extract or using a negative certified extract to build the standard calibration curve. The method of extraction consisted in acidic extraction and after a dilution is able to be measured. The analysis method results simply, achieving good detectabilities. The limits of detection (LODs) achieved were between 0.037 ± 0.01 μg kg⁻¹ in wheat, 0.71 ± 0.3 μg kg⁻¹ in barley and 0.56 ± 0.10 μg kg⁻¹ in potatoes, values that are far below the Maximum Residue Level (20 μg kg⁻¹) established by the EU policies for paraquat residues in these foodstuff products. The results demonstrate the high potential of these methods as screening tools for food safety and inspection controls.     

Validation of a method for the detection of E. coli O157:H7 in foods

This paper describes the results of a collaborative trial for the validation of a method for the detection of E. coli O157:H7 in foods. The design, sample preparation and results of the trial are discussed. An optimised laboratory protocol is proposed for improving the recovery of E. coli O157 from a range of foods which can be used as a reference method for the purposes of Official Food Control and against which other evolving methodologies can be evaluated. This protocol has also been accepted as a draft International Standard by the International Organisation for Standardisation.     

Analysis of aflatoxins in herbal medicine and health functional foods

Analytical methods of aflatoxins (AFs: B₁, B₂, G₁ and G₂) in herbal medicines (HMs) and health functional foods (HFFs) were optimized, which were used to analyze the representative samples that were directly collected from the users (total 2348 examinees) of HMs and HFFs. Two analytical methods, trifluoroacetic acid and Kobra cell derivatization methods, were compared; The latter was selected based on high linearity and sensitivity. The limits of detection of AFs using the Kobra cell method were 0.07–0.32 ng g⁻¹. Recoveries of AFs using various matrixes such as solid, semi-solid, liquid samples and CRM were 81.81–119.87%. The Z-score and linearities of calibration curves were 0.53 and 0.9996–0.9999, respectively. Among 241 samples, only Angelica gigas NAKAI extract products (2 products) were detected to have 7.93 and 5.70 ng g⁻¹ of aflatoxin G₂.     

Rapid detection of Salmonella using a redox cycling-based electrochemical method

An electrochemical method based on redox cycling combined with immunomagnetic separation and pre-concentration was developed for rapid and sensitive detection of Salmonella. Electrochemical methods for the detection of bacteria offer the advantages of instant quantification with minimal equipment. Unfortunately, the limits of detection are often poor compare to other transduction methods such as fluorescence and chemiliuminescence. We demonstrated an electrochemical method which is both rapid and has a low limit of detection. A two-step strategy, which included immunomagentic pre-concentration and redox cycling was used to amplify the signal. Magnetic beads modified with anti-Salmonella antibodies were used for separation and pre-concentration of Salmonella from phosphate buffered saline (PBS) and agricultural water. Then anti-Salmonella antibodies conjugated with alkaline phosphatase were employed for labeling the Salmonella which had been captured by magnetic beads. Alkaline phosphatase (ALP) catalyzed the substrate l-ascorbic acid 2-phosphate (AAP) to electroactive species l-ascorbic acid (AA) while tris(2-carboxyethyl)phosphine (TCEP) facilitated the regeneration of AA on the gold electrode to form redox cycling resulting in an amplified signal. Under the optimal conditions, the Salmonella in PBS buffer as well as in agricultural water were detected. The limit of detection of this approach was approximately 7.6 × 10² CFU/mL and 6.0 × 10² CFU/mL in PBS buffer and agricultural water, respectively, without pre-enrichment in 3 h. When the agricultural water has been pre-enriched for 4 h, the limit of detection was approximately 10 CFU/mL.     

采用GC-FPD法在线检测天然气中总硫含量的方法探讨

目的 通过试验研究,形成采用气相色谱-火焰光度法(GC-FPD法)在线检测总硫含量的技术。方法 比选仪器分析流程和优化分析参数,配置在线取样的降压装置和防爆外壳,并应用脱除颗粒物和液烃的集成化样品预处理技术,建立了GC-FPD法在线检测天然气中总硫含量的方法。探讨了方法的有效性、检出限、重复性和准确性等技术指标。结果 采用测定各种硫化合物含量的加和法,得到总硫含量,其中,单一硫化合物检出限为0.10 mg/m³,测量总硫含量相对标准偏差≤3%,与分析气体标准物质的比对偏差≤7%,不同方法的比对偏差≤13%。结论 采用GC-FPD技术,首次实现了天然气中13种硫化合物的在线检测,能同时检测出单一硫化合物含量和总硫含量,该方法具有较高的精密度和准确度,为在线监测天然气中总硫含量提供了可靠的技术手段。     

Cloth-based hybridization array system for the identification of Escherichia coli O157:H7

A simple cloth-based hybridization array system (CHAS) utilizing polyester cloth as the solid phase for DNA hybridizations has been adapted as a tool for the identification of presumptive verotoxigenic Escherichia coli O157:H7 colonies isolated from foods by standard culture techniques. Key indicator genes for this organism, rfbO157, fliCH7, vt1 and vt2 (encoding the O and flagellar antigenic determinants and verotoxin genes, respectively) were amplified in a multiplex PCR incorporating digoxigenin-dUTP, followed by hybridization of the amplicons with an array of specific oligonucleotide probes immobilized on polyester cloth, and subsequent immunoenzymatic assay of the bound digoxigenin label. This system includes a simple internal amplification control (IAC) to gauge PCR inhibition based on the incorporation of a primer pair with complementary 3′ ends, resulting in the generation of a unique “primer-dimer” detectable by hybridization with a specific capture probe immobilized on polyester cloth. The CHAS provided sensitive and specific detection of the E. coli O157:H7gene markers, exhibiting the expected patterns of reactivity with a panel of various target and non-target organisms.